Resistance to Acetohydroxyacid Synthase-Inhibiting Herbicides in Rice

ABSTRACT

Nucleotide sequences are disclosed that may be used to impart herbicide resistance to green plants. The sources of novel herbicide resistance were originally isolated in mutant rice plants. The sequences impart pre-emergence resistance, post-emergence resistance, or both pre-emergence resistance and post-emergence resistance to multiple herbicides. To date, resistance has been demonstrated against at least the following herbicides: imazethapyr, imazapic, imazapyr, imazamox, sulfometuron methyl, imazaquin, chlorimuron ethyl, metsulfuron methyl, rimsulfuron, thifensulfuron methyl, pyrithiobac sodium, tribenuron methyl, and nicosulfuron. Green plants transformed with these sequences are resistant to these herbicides and to derivatives of these herbicides, and to at least some of the other herbicides that normally inhibit acetohydroxyacid synthase (AHAS), particularly imidazolinone and sulfonylurea herbicides.

This is a continuation of patent application Ser. No. 11/109,587, filedApr. 19, 2005, now U.S. Pat. No. 7,399,905, issued Jul. 15, 2008; whichwas a continuation of patent application Ser. No. 10/258,842, 35 U.S.C.§ 371 date Oct. 28, 2002, now U.S. Pat. No. 6,943,280, issued Sep. 13,2005; which was the United States national stage of internationalapplication PCT/US01/15072, international filing date May 9, 2001, andpublished as WO 01/85970 A2 on Nov. 15, 2001; which claimed the benefitof the May 10, 2000 filing date of U.S. provisional application Ser. No.60/203,434 under 35 U.S.C. § 119(e); the complete disclosures of all ofwhich are hereby incorporated by reference.

TECHNICAL FIELD

This invention pertains to herbicide resistant plants, and to nucleotidesequences conferring herbicide resistance to plants, particularlyresistance to herbicides that normally interfere with the plant enzymeacetohydroxyacid synthase (AHAS), such herbicides including for examplethose of the imidazolinone class and those of the sulfonylurea class.

BACKGROUND ART

The development of novel herbicide resistance in plants offerssignificant production and economic advantages. As one example, riceproduction is frequently restricted by the prevalence of a weedyrelative of rice that flourishes in commercial rice fields. The weed iscommonly called “red rice,” and belongs to the same species ascultivated rice (Oryza sativa L.). The genetic similarity of red riceand commercial rice has made herbicidal control of red rice difficult.The herbicides Ordram™ (molinate: S-ethylhexahydro-1-H-azepine-1-carbothioate) and Bolero™ (thiobencarb:S-[(4-chlorophenyl)methyl]diethylcarbamothioate) offer partialsuppression of red rice, but no herbicide that actually controls redrice is currently used in commercial rice fields because of thesimultaneous sensitivity of existing commercial cultivars of rice tosuch herbicides. The release of mutant commercial rice lines havingresistance to herbicides that are effective on red rice will greatlyincrease growers' ability to control red rice infestations. Thedevelopment of herbicide resistance in other crops and other plants willhave similar benefits.

Rice producers in the southern United States typically rotate rice cropswith soybeans to help control red rice infestations. While this rotationis not usually desirable economically, it is frequently necessarybecause no herbicide is currently available to control red riceinfestations selectively in commercial rice crops. During the soybeanrotation, the producer has a broad range of available herbicides thatmay be used on red rice, so that rice may again be grown the followingyear. United States rice producers can lose $200-$300 per acre per yeargrowing soybeans instead of rice, a potential loss affecting about 2.5million acres annually. Additional losses in the United States estimatedat $50 million per year result from the lower price paid by mills forgrain shipments contaminated with red rice. Total economic losses due tored rice in the southern United States alone are estimated to be $500 to$750 million a year. Economic losses due to red rice may be even greaterin other rice-producing countries.

Rice producers typically use the herbicides propanil (trade name Stam™)or molinate (trade name Ordram™) to control weeds in rice production.Propanil has no residual activity. Molinate is toxic to fish. Neither ofthese herbicides controls red rice. Imazethapyr((±)-2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-5-ethyl-3-pyridinecarboxylicacid) offers an environmentally acceptable alternative to molinate, hasthe residual weed control activity that propanil lacks, and is a veryeffective herbicide on red rice. Imazethapyr also offers excellentcontrol of other weeds important in rice production, includingbarnyardgrass. Barnyardgrass is a major weed in rice production, and iscurrently controlled with propanil or molinate. However, barnyardgrasshas developed resistance to propanil in some regions.

The total potential market for rice varieties that are resistant to aherbicide that can control red rice is about 5.3 million acres in theUnited States, and the potential market outside the United States ismuch larger. World rice production occupies about 400 million acres. Redrice and other weeds are major pests in rice production in the UnitedStates, Brazil, Australia, Spain, Italy, North Korea, South Korea,Philippines, Vietnam, China, Taiwan, Brazil, Argentina, Colombia, India,Pakistan, Bangladesh, Japan, Ecuador, Mexico, Cuba, Malaysia, Thailand,Indonesia, Sri Lanka, Venezuela, Myanmar, Nigeria, Uruguay, Peru,Panama, Dominican Republic, Guatemala, Nicaragua, and otherrice-producing areas.

A number of herbicides target acetohydroxyacid synthase (AHAS), anenzyme also known as acetolactate synthase (ALS), and also designated asE.C. 4.1.3.18. This enzyme catalyzes the first step in the synthesis ofthe amino acids leucine, valine, and isoleucine. Inhibition of the AHASenzyme is normally fatal to plants. Herbicides that inhibit the enzymeacetohydroxyacid synthase would offer a number of advantages overcurrently available herbicides if they could be used in commercial riceproduction, and the production of other crops, in circumstances wherethey could not otherwise be used. Potential advantages include longresidual activity against weeds, effective control of the more importantweeds in rice production, including red rice, and relative environmentalacceptability. Even in regions where red rice is not currently aproblem, the availability of herbicide-resistant rice can have a majorinfluence on rice production practices by providing the farmer with anew arsenal of herbicides suitable for use in rice fields.

Total potential demand for resistance to AHAS-acting herbicides inplants other than rice is difficult to estimate, but could be very largeindeed.

U.S. Pat. No. 4,761,373 describes the development of mutantherbicide-resistant maize plants through exposing tissue cultures toherbicide. The mutant maize plants were said to have an altered enzyme,namely acetohydroxyacid synthase, that conferred resistance to certainimidazolinone and sulfonamide herbicides. See also U.S. Pat. Nos.5,304,732, 5,331,107, 5,718,079, 6,211,438, 6,211,439, and 6,222,100;and European Patent Application 0 154 204 A2.

Lee et al., “The Molecular Basis of Sulfonylurea Herbicide Resistance inTobacco,” The EMBO J., vol. 7, no. 5, pp. 1241-1248 (1988), describe theisolation and characterization from Nicotiana tabacum of mutant genesspecifying herbicide resistant forms of acetolactate synthase, and thereintroduction of those genes into sensitive lines of tobacco.

Saxena et al., “Herbicide Resistance in Datura innoxia,” Plant Physiol.,vol. 86, pp. 863-867 (1988) describe several Datura innoxia linesresistant to sulfonylurea herbicides, some of which were also found tobe cross-resistant to imidazolinone herbicides.

Mazur et al., “Isolation and Characterization of Plant Genes Coding forAcetolactate Synthase, the Target Enzyme for Two Classes of Herbicides,”Plant Physiol. vol. 85, pp. 1110-1117 (1987), discuss investigationsinto the degree of homology among acetolactate synthases from differentspecies.

U.S. Pat. No. 5,767,366 discloses transformed plants with geneticallyengineered imidazolinone resistance, conferred through a gene clonedfrom a plant such as a mutated Arabidopsis thaliana. See also a relatedpaper, Sathasivan et al., “Nucleotide Sequence of a Mutant AcetolactateSynthase Gene from an Imidazolinone-resistant Arabidopsis thaliana var.Columbia,” Nucleic Acids Research vol. 18, no. 8, p. 2188 (1990).

Examples of resistance to AHAS-inhibiting herbicides in plants otherthan rice are disclosed in U.S. Pat. No. 5,013,659; K. Newhouse et al.,“Mutations in corn (Zea mays L.) Conferring Resistance to ImidazolinoneHerbicides,” Theor. Appl. Genet., vol. 83, pp. 65-70 (1991); K.Sathasivan et al., “Molecular Basis of Imidazolinone HerbicideResistance in Arabidopsis thaliana var Columbia,” Plant Physiol vol. 97,pp. 1044-1050 (1991); B. Miki et al., “Transformation of Brassica napuscanola cultivars with Arabidopsis thaliana Acetohydroxyacid SynthaseGenes and Analysis of Herbicide Resistance,” Theor. Appl. Genet., vol.80, pp. 449-458 (1990); P. Wiersma et al., “Isolation, Expression andPhylogenetic Inheritance of an Acetolactate Synthase Gene from Brassicanapus,” Mol. Gen. Genet., vol. 219, pp. 413-420 (1989); J. Odell et al.,“Comparison of Increased Expression of Wild-Type and Herbicide-ResistantAcetolactate Synthase Genes in Transgenic Plants, and Indication ofPostranscriptional Limitation on Enzyme Activity,” Plant Physiol., vol.94, pp. 1647-1654 (1990); published international patent application WO92/08794; U.S. Pat. No. 5,859,348; published international patentapplication WO 98/02527; published European Patent Application EP 0 965265 A2, and published international patent application WO 90/14000.

U.S. Pat. Nos. 5,853,973 and 5,928,937 disclose the structure-basedmodeling of AHAS, the presumptive binding pockets on the enzyme forAHAS-acting herbicides, and certain designed AHAS mutations to conferherbicide residence. These patents also disclose amino acid sequencesfor the AHAS enzymes and isozymes from several plants, including thatfrom Zea mays. See also published international patent application WO96/33270.

S. Sebastian et al., “Soybean Mutants with Increased Tolerance forSulfonylurea Herbicides,” Crop. Sci., vol. 27, pp. 948-952 (1987)discloses soybean mutants resistant to sulfonylurea herbicides. See alsoU.S. Pat. No. 5,084,082.

K. Shimamoto et al., “Fertile Transgenic Rice Plants Regenerated fromTransformed Protoplasts,” Nature, vol. 338, pp. 274-276 (1989) disclosesa genetic transformation protocol in which electroporation ofprotoplasts was used to transform a gene encoding β-glucuronidase intorice.

T. Terakawa et al., “Rice Mutant Resistant to the Herbicide BensulfuronMethyl (BSM) by in vitro Selection,” Japan. J. Breed., vol. 42, pp.267-275 (1992) discloses a rice mutant resistant to a sulfonylureaherbicide, derived by selective pressure on callus tissue culture.Resistance was attributed to a mutant AHAS enzyme.

Following are publications by the inventor (or the inventor and otherauthors) concerning research on herbicide-resistant rice varieties.These publications are T. Croughan et al., “Rice and Wheat Improvementthrough Biotechnology,” 84th Annual Research Report, Rice ResearchStation, 1992, pp. 100-103 (1993); T. Croughan et al., “Rice and WheatImprovement through Biotechnology,” 85th Annual Research Report, RiceResearch Station, 1993, pp. 116-156 (1994); T. Croughan, “Application ofTissue Culture Techniques to the Development of Herbicide ResistantRice,” Louisiana Agriculture, vol. 37, no. 3, pp. 25-26 (1994); T.Croughan et al., “Rice Improvement through Biotechnology,” 86th AnnualResearch Report, Rice Research Station, 1994, pp. 461-482 (1995); T.Croughan et al., “Assessment of Imidazolinone-Resistant Rice,” 87thAnnual Research Report, Rice Research Station, 1994, pp. 491-525(September 1996); T. Croughan et al., “IMI-Rice Evaluations,” 88thAnnual Research Report, Rice Research Station, 1996, pp. 603-629(September 1997); T. Croughan et al., “Rice Biotechnology Research,”89th Annual Research Report, Rice Research Station, 1997, p. 464(September 1998); T. Croughan et al., “Imidazolinone-Resistant Rice,”90th Annual Research Report, Rice Research Station, 1998, p. 511(December 1999); T. Croughan et al., “Rice and Wheat Improvement throughBiotechnology,” USDA CRIS Report Accession No. 0150120 (for Fiscal Year1994-actual publication date currently unknown); T. Croughan,“Improvement of Lysine Content and Herbicide Resistance in Rice throughBiotechnology,” USDA CRIS Report Accession No. 0168634 (for Fiscal Year1997-actual publication date currently unknown); T. Croughan,“Improvement of Lysine Content and Herbicide Resistance in Rice throughBiotechnology,” USDA CRIS Report Accession No. 0168634 (for Fiscal Year1999-actual publication date currently unknown); T. Croughan,“Improvement of Lysine Content and Herbicide Resistance in Rice throughBiotechnology,” USDA CRIS Report Accession No. 0168634 (for Fiscal Year2000-actual publication date currently unknown); T. Croughan, “HerbicideResistant Rice,” Proc. 25th Rice Tech. Work. Groups, p. 44 (1994); T.Croughan et al, “Applications of Biotechnology to Rice Improvement,”Proc. 25th Rice Tech. Work. Groups, pp. 62-63 (1994); T. Croughan,“Production of Rice Resistant to AHAS-Inhibiting Herbicides,” Congresson Cell and Tissue Culture, Tissue Culture Association, In Vitro, vol.30A, p. 60, Abstract P-1009 (Jun. 4-7, 1994). (Note that the AnnualResearch Reports of the Rice Research Station are published in the yearafter the calendar year for which activities are reported. For example,the 84th Annual Research Report, Rice Research Station, 1992,summarizing research conducted in 1992, was published in 1993.) Thereports in the 87th and 88th Annual Research Report, Rice ResearchStation (published September 1996 and September 1997, respectively)mention the breeding line 93AS3510 in tables giving data on certainherbicide resistance trials. These reports gave no information on howthe breeding line was developed. The breeding line was not publiclyavailable at the times these reports were published. The breeding line93AS3510 is the same as the ATCC 97523 rice that is described in greaterdetail in the present inventor's later-published international patentapplication WO 97/41218 (1997) and U.S. Pat. Nos. 5,736,629, 5,773,704,5,952,553, and 6,274,796.

See also E. Webster et al., “Weed Control Systems for Imi-Rice,” p. 33in Program of the 27th Rice Technical Working Group Meeting (March1998); L. Hipple et al., “AHAS Characterization of ImidazolinoneResistant Rice,” pp. 45-46 in Program of the 27th Rice Technical WorkingGroup Meeting (March 1998); W. Rice et al., “Delayed Flood for RiceWater Weevil Control using Herbicide Resistant Germplasm,” p. 61 inProgram of the 27th Rice Technical Working Group Meeting (March 1998);E. Webster et al., “Weed Control Systems for Imidazolinone-Rice,” p. 215in Proceedings of the 27th Rice Technical Working Group Meeting (1999);L. Hipple et al., “AHAS Characterization of Imidazolinone ResistantRice,” pp. 68-69 in Proceedings of the 27th Rice Technical Working GroupMeeting (1999); W. Rice et al., “Delayed Flood for Rice Water WeevilControl using Herbicide Resistant Germplasm,” p. 134 in Proceedings ofthe 27th Rice Technical Working Group Meeting (1999); and W. Rice etal., “Delayed flood for management of rice water weevil (Coleopterae:Curculionidae),” Environmental Entomology, vol. 28, no. 6, pp. 1130-1135(December 1999).

The present inventor's U.S. Pat. No. 5,545,822 discloses a line of riceplants having a metabolically-based resistance to herbicides thatinterfere with the plant enzyme acetohydroxyacid synthase; i.e., theherbicide resistance of these rice plants was not due to a resistantAHAS enzyme. (See published international patent application WO97/41218, pages 6-9.) See also the present inventor's U.S. Pat. No.5,773,703.

The present inventor's published international patent application WO97/41218 discloses one line of rice plants having a mutant AHAS enzymethat is resistant to herbicides that interfere with the wild-type plantenzyme acetohydroxyacid synthase. This line of rice plants was developedby exposing rice seeds to the mutagen methanesulfonic acid ethyl ester(EMS), and screening millions of progeny for herbicide resistance. Seealso the present inventor's U.S. Pat. Nos. 5,736,629, 5,773,704,5,952,553, and 6,274,796.

The present inventor's published international patent application WO00/27182 discloses additional lines of rice plants having mutant AHASenzymes that are resistant to herbicides that interfere with thewild-type plant enzyme acetohydroxyacid synthase. These lines of riceplants were developed by exposing rice seeds to EMS, and screeningmillions of progeny for herbicide resistance.

U.S. Pat. No. 4,443,971 discloses a method for preparing herbicidetolerant plants by tissue culture in the presence of herbicide. U.S.Pat. No. 4,774,381 discloses sulfonylurea (sulfonamide)herbicide-resistant tobacco plants prepared in such a manner.

U.S. Pat. No. 5,773,702 discloses sugar beets with a resistant mutantAHAS enzyme, derived from cell cultures grown in the presence ofherbicide. See also published international patent application WO98/02526.

Published international patent application WO 00/26390 discloses thecloning and sequencing of the Arabidopsis AHAS small subunit protein,and an expression vector to transform plants with that small AHASsubunit to impart herbicide tolerance.

U.S. Pat. No. 5,633,437 discloses a herbicide resistant AHAS enzyme andgene isolated from cockleburs.

U.S. Pat. No. 5,767,361 discloses a mutant, resistant AHAS enzyme frommaize. The definitions of the U.S. Pat. No. 5,767,361 are incorporatedinto the present disclosure by reference, to the extent that thosedefinitions are not inconsistent with the present disclosure, as arethat patent's descriptions of certain genetic transformation techniquesfor plants. See also U.S. Pat. No. 5,731,180 and European PatentApplication 0 525 384 A2.

U.S. Pat. No. 5,605,011; European Patent Application 0 257 993 A2; andEuropean Patent Application 0 730 030 A1 disclose resistant acetolactatesynthase enzymes derived from callus culture of tobacco cells in thepresence of herbicide, from spontaneous mutations of the ALS gene inyeast; EMS-induced mutations in Arabidopsis seeds; certain modificationsof those enzymes; and the transformation of various plants with genesencoding the resistant enzymes. These patents disclose severaltechniques for modifying AHAS genes to produce herbicide-resistant AHASenzymes, and for transforming plants with those genes.

U.S. Pat. Re No. 35,661 (a reissue of U.S. Pat. No. 5,198,599) discloseslettuce plants with enhanced resistance to herbicides that target theenzyme acetolactate synthase. The initial source of herbicide resistancewas a prickly lettuce weed infestation in a grower's field, aninfestation that was not controlled with commercial sulfonylureaherbicides.

T. Shimizu et al., “Oryza sativa ALS mRNA for acetolactate synthase,complete cds, herbicide sensitive wild type,” BLAST accession numberAB049822 (April 2001), available through www.ncbi.nlm.nih.gov/blast,discloses the nucleotide sequence and inferred amino acid sequence forwild type ALS cDNA from Oryza sativa var. Kinmaze. These two sequencesare reproduced below as SEQ ID NOS 2 and 3, respectively.

T. Shimizu et al., “Oryza sativa ALS mRNA for acetolactate synthase,complete cds, herbicide resistant biotype,” BLAST accession numberAB049823 (April 2001), available through www.ncbi.nlm.nih.gov/blast,discloses the nucleotide sequence and inferred amino acid sequence foran ALS cDNA from Oryza sativa var. Kinmaze that was reported to beherbicide resistant, although the nature of the herbicide resistance isnot specified in the BLAST description. These two sequences arereproduced below as SEQ ID NOS 4 and 5, respectively.

Following are selected data taken from some of the references citedabove, concerning the locations of certain imidazolinone or sulfonylureaherbicide tolerance mutations in AHAS/ALS from various species. Noattempt has been made to reconcile or align the different nucleotide oramino acid numbering systems used in the different references. Indescribing the substitutions below (as well as in the remainder of thespecification and the claims), the wild-type nucleotide or amino acid isalways listed first, followed by the mutant nucleotide or amino acid.All substitutions discussed refer to the AHAS/ALS DNA coding sequence,or to the expressed or inferred AHAS/ALS amino acid sequence.

Lee et al. (1988) reported that there were two homologous ALS genes inNicotiana tabacum. The sulfonylurea herbicide-resistant C3 mutant in oneALS gene had a Pro-Gln replacement at amino acid 196; while thesulfonylurea herbicide-resistant S4-Hra mutant in the other ALS gene hadtwo amino acid changes: Pro-Ala at amino acid 196, and Trp-Leu at aminoacid 573.

Sathasivan et al. (1990), Sathasivan et al. (1991), and U.S. Pat. No.5,767,366 reported a G-A nucleotide substitution at position 1958,corresponding to a Ser-Asn substitution at position 653, in animidazolinone herbicide-resistant Arabidopsis thaliana.

Wiersma et al. (1989) reported sulfonylurea herbicide resistance intobacco plants that had been transformed with a mutant Brassica napusALS gene, in which codon 173 had been altered by site-directedmutagenesis to replace Pro with Ser.

European patent application 0 257 993 A2 reported several spontaneousmutations in the yeast (Saccharomyces cerevisiae) ALS gene that resultedin sulfonylurea herbicide resistance: at amino acid position 121, asubstitution of wild-type Gly by Ser; at amino acid position 122, asubstitution of wild-type Ala by Pro, Asp, Val, or Thr; at position 197,a substitution of wild-type Pro by Ser or Arg; at position 205, asubstitution of wild-type Ala by Asp or Thr; at position 256, asubstitution of wild-type Lys by Glu, Thr, or Asn; at position 359, asubstitution of wild-type Met by Val; at position 384, a substitution ofwild-type Asp by Glu, Val, or Asn; at position 588, a substitution ofwild-type Val by Ala; at position 591, a substitution of wild-type Trpby Arg, Cys, Gly, Leu, Ser, or Ala; at position 595, a substitution ofwild-type Phe by Leu. The same patent application reported severalsite-directed mutations of the yeast ALS gene at some of the samepositions to also produce sulfonylurea herbicide resistance: at aminoacid position 122, a substitution of wild-type Ala by Ser, Val, Thr,Pro, Asn, Ile, His, Arg, Leu, Tyr, Cys, Phe, Glu, Met, Lys, Gln, or Trp;at position 205, a substitution of wild-type Ala by Arg, Cys, Glu, orTrp; at position 256, a substitution of wild-type Lys by Asp, Gly, Leu,Pro, or Trp; at position 359, a substitution of wild-type Met by Pro orGlu; at position 384, a substitution of wild-type Asp by Pro, Trp, Ser,Gly, Cys, or Lys; at position 591, a substitution of wild-type Trp byAsp, Glu, Phe, His, Tyr, Ile, Val, Lys, Arg, Met, Asn, Gln, or Thr. Seealso U.S. Pat. No. 5,605,011, which also describes experimental data forthe following site-directed mutations: at amino acid 121, a substitutionof wild-type Gly by Asn or Ala; at amino acid 197, a substitution ofwild-type Pro by Gln, Glu, Ala, Gly, Trp, Tyr, Cys, or Val; at aminoacid 205, a substitution of wild-type Ala by Tyr, Val, or Asn; at aminoacid 359, a substitution of wild-type Met by Gln, Lys, Tyr, or Cys; atposition 583, a substitution of wild-type Val by Ser, Asn, Trp, or Cys;and at position 595, a substitution of wild-type Phe by Gly, Asn, Arg,Cys, Pro, Ser, or Trp. Other amino substitutions at the same positionsare also described prophetically, without experimental data. See alsoU.S. Pat. No. 5,013,659.

WO 98/02527 reported sulfonylurea and triazolopyrimidine resistance inone line of sugar beets resulting from a C-T substitution at nucleotide562, corresponding to a Pro-Ser substitution at amino acid 188. Thissame reference also reported sulfonylurea, imidazolinone, andtriazolopyrimidine resistance in a second line of sugar beets resultingfrom two mutations: The same mutation as reported in the first line(from which the second line had been derived), coupled with a G-Asubstitution at nucleotide 337, corresponding to an Ala-Thr substitutionat amino acid 113. See also WO 98/02526, U.S. Pat. Nos. 5,859,348 and5,773,702.

WO 96/33270 describes a number of designed or predicted mutations from astructure-based modeling method, that were said to induce imidazolinonetolerance in AHAS Experimental results confirming such tolerance inmutated Arabidopsis AHAS, either in vitro or in transformed tobaccoplants in vivo were provided for the following substitutions: Met-Ile atamino acid position 124, Met-His at position 124, Arg-Glu at position199, and Arg-Ala at position 199. See also U.S. Pat. Nos. 5,928,937 and5,853,973.

WO 92/08794 reported imidazolinone resistance in two lines of maize. Onehad a G-A substitution at nucleotide position 171, resulting in anAla-Thr substitution at the corresponding amino acid position. The otherhad a G-A substitution at position 1888, resulting in a Ser-Asnsubstitution at the corresponding amino acid position.

U.S. Pat. No. 5,731,180 reported imidazolinone resistance in maizeresulting from a G-A substitution at nucleotide position 1898, resultingin a Ser-Asn substitution at amino acid position 621. See also U.S. Pat.No. 5,767,361 and European patent application 0 525 384.

U.S. Pat. No. 5,633,437 reported imidazolinone resistance in cockleburs,characterized by five differences between resistant ALS enzyme biotypesand sensitive biotypes: Lys-Glu at amino acid position 63, Phe-Leu atposition 258, Gln-His at position 269, Asn-Ser at position 522, andTrp-Leu at position 552. The changes at positions 522 and 552 werethought to be particularly important.

T. Shimizu et al., “Oryza sativa ALS mRNA for acetolactate synthase,complete cds, herbicide resistant biotype,” BLAST accession numberAB049823 (April 2001) reported a nucleotide sequence and inferred aminoacid sequence for a rice ALS that was said to be herbicide resistant,although the nature of the herbicide resistance was not specified in theBLAST entry. As compared to a contemporaneous wild type ALS for the samerice variety (Kinmaze), the inferred amino acid sequence for theresistant ALS appeared to display two differences: a Trp-Leusubstitution at position 548, and a Ser-Ile substitution at position627.

DISCLOSURE OF INVENTION

I have discovered nucleotide sequences that may be used to impartherbicide resistance to green plants. These sources of novel herbicideresistance were originally isolated in mutant rice plants. Thenucleotide sequences impart pre-emergence resistance, post-emergenceresistance, or both pre-emergence resistance and post-emergenceresistance to multiple herbicides. To date, resistance has beendemonstrated against at least the following herbicides, as well as somemixtures of the same herbicides: imazethapyr, imazapic, imazapyr,imazamox, sulfometuron methyl, imazaquin, chlorimuron ethyl, metsulfuronmethyl, rimsulfuron, thifensulfuron methyl, pyrithiobac sodium,tribenuron methyl, and nicosulfuron. Green plants transformed with thesenucleotide sequences are also resistant to derivatives of theseherbicides, and to at least some of the other herbicides that normallyinhibit acetohydroxyacid synthase (AHAS), particularly imidazolinone andsulfonylurea herbicides. The degree of herbicide resistance imparted bysome of the novel nucleotide sequences is comparable to (and is possiblygreater than) the highest levels of resistance to AHAS-inhibitingherbicides that have previously been found in resistant mutants of anyspecies of green plant that is normally susceptible to this class ofherbicides.

These nucleotide sequences may be used to transform green plantsincluding, but not limited to, rice. Alternatively, analogous mutationsmay be introduced into green plants by site-directed mutagenesis of theplant's native AHAS coding sequence(s). As a particular example,transformation of the nucleotide sequences into rice plants, orsite-directed mutagenesis of rice plants, will accelerate what couldalso be accomplished, perhaps more slowly, by traditional breedingtechniques such as crossing and back-crossing. Since no coding sequencefrom another species is thereby introduced into the rice, mostresearchers would not consider such a transformed rice plant to be“transgenic.” (Not even a marker gene is needed for such atransformation, since selection may be performed directly for theherbicide resistance trait itself.) Similarly, where site-directedmutagenesis is used to introduce an analogous point mutation into thenative AHAS coding sequence of a green plant other than rice, mostresearchers would not consider the resulting plant to be transgenic.

Besides controlling red rice, many AHAS-inhibiting herbicides alsoeffectively control other weeds commonly found in fields in which riceand other crops are grown. Several of these herbicides have residualactivity, so that one treatment controls both existing weeds and weedsthat sprout later—a significant advantage in production.

No herbicide currently labelled for use on rice has residual activityagainst a broad spectrum of weeds including red rice. With effectiveresidual activity against red rice and other weeds, rice producers nowhave a weed control system far superior to those currently used. Onerole of water in rice production is in weed control—a layer of standingwater in the rice field inhibits the growth of weeds. With a herbicidehaving residual weed control properties, producers will have muchgreater flexibility in water management. Flooding of fields may now bedelayed, which in turn will help control the rice water weevil, aprimary insect pest of rice. Alternatively, or perhaps in conjunction,pumping costs could be reduced by delaying flooding until sufficientrain falls to flood a field at no cost to the producer.

The herbicide resistance of each of the novel nucleotide sequences isattributable to mutation of the expressed rice AHAS enzyme, producingenzymes that express direct resistance to levels of herbicide thatnormally inhibit the wild-type AHAS enzymes. That the resistance is dueto mutant AHAS enzymes (rather than another route such as gene copynumber, enhanced promoter activity, metabolic degradation, etc.) hasbeen confirmed by in vitro assays.

The following mutations have been observed in one or more of the AHAScoding sequences from the various herbicide-resistant rice lines: (1)Some of the herbicide-resistant lines have a serine-to-asparaginemutation in the codon corresponding to amino acid location 627. (2) Oneof the herbicide-resistant lines is believed to have a serine-to-lysinemutation in the codon corresponding to amino acid location 627, coupledwith a deletion causing a frame shift, leading to a stop codon soonthereafter.

Some of the mutant rice AHAS amino acid sequences reported here show aserine-to-asparagine mutation at amino acid 627, a location near thecarboxy terminus of the AHAS protein that is analogous to the locationof serine-to-asparagine mutations that have been previously reported inimidazolinone herbicide-resistant AHAS from Arabidopsis and maize plants(see discussion above). However, the novel sequences show surprisingproperties that could not have been predicted from the earlier work. Thedegree of herbicide resistance imparted by some of the novel nucleotidesequences is comparable to (and is possibly greater than) the highestlevels of resistance to AHAS-inhibiting herbicides that have previouslybeen found in resistant mutants of any species of green plant that isnormally susceptible to this class of herbicides. The underlying reasonis currently unknown. Without wishing to be bound by this theory, it ispossible that the rice AHAS molecule provides a particularly favorablesetting for the Ser-Asn mutation. In other words, it is hypothesizedthat, as compared to other plants in which a Ser-Asn AHAS mutation haspreviously been reported, there exists a synergy between other portionsof the rice AHAS molecule and the site of the mutation, leading to somevery high levels of herbicide resistance. These very high levels ofherbicide resistance make the novel mutant rice AHAS sequences moreattractive as candidates for transforming other plants for herbicideresistance.

From the data reported here, as well as data reported in some of thereferences previously cited, it appears that mutations at locationshomologous to the 627 serine in the rice AHAS molecule can lead toresistance to AHAS-inhibiting herbicides in green plants. Withoutwishing to be bound by this theory, it is hypothesized that a Ser-Asnamino substitution at this location is particularly favorable for aherbicide resistant phenotype. We also report results here for lineCMC31 showing instead a Ser-Lys substitution at this location, followedby a frame-shift mutation; and we note that the Shimizu (April 2001)BLAST submissions reported herbicide resistance (of currently unknownnature) following a Ser-Ile substitution at position 627 and a Trp-Leusubstitution at position 548.

Due to their chemical similarity to Asn, their steric similarity to Asn,or both, it is also expected that the following amino acid substitutionsat locations homologous to the 627 serine in the rice AHAS molecule willalso lead to resistance to AHAS-inhibiting herbicides: Gln, Asp, andGlu. These mutations could not, however, result from single-nucleotidesubstitutions in the coding sequence, and would therefore beconsiderably less likely to result from undirected mutation breedingefforts. However, these mutations could be induced by site-directedmutagenesis techniques known in the art. See, e.g., R. Higuchi,“Recombinant PCR,” pp. 177-183 in M. Innis et al. (Eds.), PCR Protocols:A Guide to Methods and Applications, Academic Press (1990); U.S. Pat.No. 6,010,907; Kunkel, Proc. Natl. Acad. Sci. USA, vol. 82, pp. 488-492(1985); Kunkel et al., Methods Enzymol., vol. 154, pp. 367-382 (1987);U.S. Pat. No. 4,873,192; Walker et al. (Eds.), Techniques in MolecularBiology (MacMillan, New York, 1983); or the Genoplasty™ protocols ofValiGen (Newtown, Pa.).

For that matter, any of the mutations described here may be incorporatedinto the genome of rice or of any other green plant using site-directedmutagenesis. Doing so can, among other things, speed the process ofintroducing herbicide resistance into an existing cultivar, byeliminating the time needed for crossing and back-crossing throughtraditional breeding techniques, and without introducing a gene fromanother species.

Likewise, the Ser-Lys substitution at position 627, followed by a frameshift could instead be a Ser-Arg or Ser-His substitution, followed by aframe shift, since Lys, Arg, and His are chemically similar.

Thus one aspect of this invention provides green plants transformed withan oligonucleotide sequence encoding the rice AHAS molecule, in whichthe serine at amino acid position 627 has been replaced with asparagine.

Another aspect of this invention provides green plants having anoligonucleotide sequence encoding an AHAS molecule in which the serinehomologous to that of position 627 in the rice AHAS molecule has beenreplaced with glutamine, glutamic acid, or aspartic acid. The AHASmolecule may otherwise be native to the same plant in which it is beingexpressed, or it may be derived from another plant. One embodiment ofthis aspect of the invention is a green plant comprising anoligonucleotide sequence encoding an AHAS molecule identical to thewild-type rice AHAS molecule, except that the serine of position 627 inthe rice AHAS molecule has been replaced with glutamine, glutamic acid,or aspartic acid. If the mutant AHAS molecule is otherwise native to theplant in which it is expressed, except for the substitution at position627, then such a plant should not be considered a “genetically modifiedorganism,” in the popular sense of an organism that has beenartificially transformed with an oligonucleotide coding sequence derivedfrom a different species. For example, such a plant could be a riceplant containing an oligonucleotide sequence encoding an AHAS moleculeidentical to the wild-type rice AHAS molecule, except that the serine ofposition 627 in the rice AHAS molecule has been replaced with glutamine,glutamic acid, or aspartic acid.

Another aspect of this invention provides green plants having anoligonucleotide sequence encoding an AHAS molecule in which the serinehomologous to that of position 627 in the rice AHAS molecule has beenreplaced with lysine, arginine, or histidine—preferably lysine—followedby whatever amino acids may be encoded between position 627 and thecarboxy terminus as the result of a frame shift in the AHAS codingsequence at or following position 627. The AHAS molecule may otherwisebe native to the same plant in which it is being expressed, or it may bederived from another plant. One embodiment of this aspect of theinvention is a green plant comprising an oligonucleotide sequenceencoding an AHAS molecule identical to the wild-type rice AHAS molecule,except that the serine of position 627 in the rice AHAS molecule hasbeen replaced with lysine, arginine, or histidine—preferablylysine—followed by whatever amino acids may be encoded between position627 and the carboxy terminus as the result of a frame shift in the AHAScoding sequence at or following position 627. If the mutant AHASmolecule is otherwise native to the plant in which it is expressed,except for the substitutions at position 627 and subsequent, then such aplant should not be considered a “genetically modified organism,” in thepopular sense of an organism that has been artificially transformed withan oligonucleotide coding sequence derived from a different species. Forexample, such a plant could be a rice plant containing anoligonucleotide sequence encoding an AHAS molecule identical to thewild-type rice AHAS molecule, except that the serine of position 627 inthe rice AHAS molecule has been replaced with lysine, arginine, orhistidine—preferably lysine—followed by whatever amino acids may beencoded between position 627 and the carboxy terminus as the result of aframe shift in the AHAS coding sequence at or following position 627.

The procedures used below to assay the activity of the acetohydroxyacidsynthases were substantially as described in B. K. Singh et al., “Assayof Acetohydroxyacid Synthase,” Analytical Biochemistry, vol. 171, pp.173-179 (1988), except as noted. In the first paragraph of Singh's“Materials and Methods,” instead of corn suspension culture cells, shoottissues from greenhouse-grown rice seedlings at the 3-4 leaf stage ofdevelopment, or rice suspension culture cells were used. For shoottissues, 40.0 grams (fresh weight) of tissue were extracted in the samemanner for each of the breeding lines; for Cypress suspension cells,16.0 grams of cells were used, harvested eight days after subculture. Atthe suggestion of the first author, B. K. Singh (personalcommunication), the desalting step mentioned at the bottom of Singh'sfirst column under “Materials and Methods” was eliminated. Pursuit™herbicide (imazethapyr) or Arsenal™ herbicide (imazapyr) was included inthe “standard reaction mixture” for the AHAS assay in variousconcentrations. Colorimetric absorbance was measured at 520 nm. Checkswere made of direct acetoin formation during the enzyme assay.

An alternative AHAS assay that could be used (but that was not used incollecting the data reported here) is that disclosed in U.S. Pat. No.5,605,011, at col. 53, line 61 through col. 54, line 37.

MODES FOR CARRYING OUT THE INVENTION

A total of 27 new rice lines expressing resistance to AHAS-inhibitingherbicides were identified, following exposure of rice seeds to themutagen methanesulfonic acid ethyl ester (EMS). Additional resistantrice lines will be developed and identified using similar mutation andscreening techniques. Other mutagens known in the art may be substitutedfor EMS in generating such mutations, for example, sodium azide,N-methyl-N-nitrosourea, N-ethyl-N-nitrosourea, nitrosoquanidine,hydroxylamine, hydrazine, ionizing radiation (such as X-rays, gammarays, or UV), or radiomimetic compounds such as bleomycins, etoposide,and teniposide. (Bleomycins, for example, are glycopeptide antibioticsisolated from strains of Streptomyces verticillus. One bleomycin is soldunder the trademark Blenoxane® by Bristol Laboratories, Syracuse, N.Y.)The resistant AHAS nucleotide sequences from these rice lines will becloned and used to transform other rice lines, and lines of other greenplants, to impart herbicide resistance characteristics.

EXAMPLES 1-15

Approximately 52 million mutated (M₂) rice seed were screened. Themutated seed were developed by soaking a total of 340 pounds of seed(M₁), of the rice cultivars “Cypress” or “Bengal,” in a 0.175% (byweight) aqueous solution of EMS. Approximately 170 lb. of rice wereexposed to EMS for 16 hours; approximately 85 lb. were exposed for 24hours; and approximately 85 lb were exposed for 35 hours. Seed from thethree exposure regimens were pooled for the screening experimentsdescribed below.

Following EMS treatment, the M₁ seed were thoroughly rinsed with waterand drained before being planted by broadcast-seeding into shallowwater, water that was drained 24 hours later. The field was re-floodedthree days later, and the field was maintained in a flooded conditionuntil it was drained for harvesting. The harvested M₂ seed were storedover the winter, and plants grown from the M₂ seed were screened forherbicide resistance the following spring. Following drill-seeding ofapproximately 52 million M₂ seed, a pre-emergence application ofimazethapyr at a rate of 0.125 lb ai/A (pounds of active ingredient peracre) was applied prior to the first flush. A post-emergence treatmentof imazethapyr at 0.063 lb ai/A was applied when the rice reached the3-leaf stage. The fifteen M₂ plants that survived the herbicideapplication were collected and transferred to the greenhouse.

The herbicide resistance of the progeny of these plants (M₃) wasconfirmed through a post-emergence application of 0.125 lb ai/Aimazethapyr at the 3-leaf stage in the greenhouse. The 15 resistantplants of 52 million total M₂ plants represent a success rate ofapproximately 1 imidazolinone-resistant mutant identified per 3.5million mutated seeds screened.

M₄ progeny seed were collected from the resistant M₃ plants, and wereused in a field test. The field test comprised 8 replicate sets. Each ofthe sets contained 100 rows, each row four feet in length. Each of thesets had 74 rows of the M₄ resistant lines. Each set had multiple rowsof each of the 15 resistant lines, with the number of rows of each ofthe lines varying due to the different numbers of seeds of each thatwere available at the time. Each of the replicate sets also contained 16rows of the non-resistant cultivar “Cypress” as a negative control, and10 rows of earlier-developed herbicide-resistant rice lines as positivecontrols. (The positive controls were either ATCC 97523 or a hybrid ofATCC 97523 and ATCC 75295.) A different herbicide treatment was appliedpost-emergence to each of these eight replicate sets when the ricereached the 3-leaf stage. The control set was treated with 4 quarts/acreof Arrosolo™. Arrosolo™ is a herbicide that is currently usedcommercially with conventional rice varieties. The remaining 7 sets weretreated with imidazolinone herbicides as follows: (1) imazethapyr (tradename Pursuit™ or Newpath™) at 0.125 lb ai/A; (2) imazethapyr at 0.188 lbai/A; (3) imazapic (trade name Cadre™) at 0.063 lb ai/A; (4) imazapic at0.125 lb ai/A; (5) imazapyr (trade name Arsenal™) at 0.05 lb ai/A; (6)imazapyr at 0.09 lb ai/A; and (7) a mixture of 75% imazethapyr and 25%imazapyr (trade name Lightning™) at 0.052 lb ai/A.

Note that all herbicide application rates tested were equal to orgreater than the recommended application rates for the use of the sameherbicides on other crops.

Levels of resistance to herbicide were determined both at three weeksafter spraying, and at maturity. No row was significantly injured by thecontrol treatment with the conventional rice herbicide Arrosolo™. Bycontrast, each of the seven imidazolinone treatments resulted in 100%control of the rows of non-resistant Cypress rice, without a singlesurviving plant among any of the 112 treated rows. Each of theherbicide-resistant M₄ progeny rows in each of the sets, and each of theherbicide-resistant positive controls in each of the sets, displayedinsignificant injury or no injury from the various imidazolinonetreatments. The rows of resistant M₄ progeny treated with theimidazolinones, and the rows of herbicide-resistant positive controlstreated with the imidazolinones, were visually indistinguishable fromthe Arrosolo™-treated rows with respect to height, vigor, days tomaturity, and lack of visible herbicide injury.

Samples of the seed harvested from each of the fifteen lines of the M₄progeny, i.e., samples of M₅ seed from each of the fifteen separatelines; lines designated by the inventor as SSC01, SSC02, SSC03, SSC04,SSC05, SSC06, SSC07, SSC08, SSC09, SSC10, SSC11, SSC12, SSC13, SSC14,and SSC15; were separately deposited with the American Type CultureCollection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209on 5 Nov. 1998; and were assigned ATCC Accession Nos. 203419, 203420,203421, 203422, 203423, 203424, 203425, 203426, 203427, 203428, 203429,203430, 203431, 203432, and 203433, respectively. Each of these depositswas made pursuant to a contract between ATCC and the assignee of thispatent application, Board of Supervisors of Louisiana State Universityand Agricultural and Mechanical College. Each of the contracts with ATCCprovides for permanent and unrestricted availability of these seeds orthe progeny of these seeds to the public on the issuance of the U.S.patent describing and identifying the deposit or the publication or thelaying open to the public of any U.S. or foreign patent application,whichever comes first, and for the availability of these seeds to onedetermined by the U.S. Commissioner of Patents and Trademarks (or by anycounterpart to the Commissioner in any patent office in any othercountry) to be entitled thereto under pertinent statutes andregulations. The assignee of the present application has agreed that ifany of the seeds on deposit should become nonviable or be lost ordestroyed when cultivated under suitable conditions, they will bepromptly replaced on notification with a viable sample of the sameseeds.

EXAMPLES 16-27

Approximately 60 million additional mutated (M₂) rice seed werescreened. The mutated seed were developed by soaking a total of 300pounds of seed (M₁) of the rice cultivar “Cypress” in a 0.175% (byweight) aqueous solution of the mutagen EMS for 23 hours.

Following EMS treatment the M₁ seed were thoroughly rinsed with waterand drained before being planted by broadcast-seeding into shallowwater, water that was drained 24 hours later. The field was re-floodedthree days later, and the field was maintained in a flooded conditionuntil it was drained for harvesting. The harvested M₂ seed were storedover the winter, and plants grown from the M₂ seed were screened forherbicide resistance the following spring. Following broadcast-seedingand shallow soil incorporation of approximately 60 million M₂ seed, apost-emergence application of imazapic (trade name Cadre™) at 0.125 lbai/A was sprayed on half the field, and a post-emergence application ofimazapyr (trade name Arsenal™) at 0.10 lb ai/A was applied to theremaining half of the field at the three-leaf stage. The twenty-three M₂plants that survived the herbicide application were collected andtransferred to the greenhouse. Later testing (described below) showedthat twelve of these plants represented new herbicide resistant lines;the other plants were “volunteer” seed of the ATCC 97523 line that hadremained in the soil from a prior season.

The 12 resistant plants of 60 million total M₂ plants represent asuccess rate of approximately 1 imidazolinone-resistant mutantidentified per 5 million mutated seeds screened.

The herbicide resistance of the progeny of these plants (M₃) wasconfirmed with the following herbicide applications in the greenhouse:0.125 lb ai/A imazethapyr (trade name Pursuit™) as a pre-emergenceapplication; 0.063 lb ai/A imazethapyr as a post-emergence application;0.10 lb ai/A sulfometuron methyl (trade name Oust™) as a pre-emergenceapplication; 0.05 lb ai/A sulfometuron methyl as a post-emergenceapplication; 0.10 lb ai/A nicosulfuron (trade name Accent™) appliedpre-emergence; and 0.05 lb ai/A nicosulfuron applied post-emergence. TwoM₃ seed from each of the twenty-three herbicide-resistant lines wereplanted in each of four replicate pots for each treatment. Equivalentplantings of control lines were made with (non-resistant) Cypress andBengal rice seeds.

Samples of the seed harvested from several of these lines of the M₄progeny; namely, samples of M₅ seed from each of the seven separatelines designated by the inventor as PWC16, PWC23, CMC29, CMC31, WDC33,WDC37, and WDC38; were separately deposited with the American TypeCulture Collection (ATCC), 10801 University Boulevard, Manassas, Va.20110-2209 on 2 Nov. 1999; and were assigned ATCC Accession Nos.PTA-904, PTA-905, PTA-902, PTA-903, PTA-906, PTA-907, and PTA-908,respectively. Each of these deposits was made pursuant to a contractbetween ATCC and the assignee of this patent application, Board ofSupervisors of Louisiana State University and Agricultural andMechanical College. Each of the contracts with ATCC provides forpermanent and unrestricted availability of these seeds or the progeny ofthese seeds to the public on the issuance of the U.S. patent describingand identifying the deposit or the publication or the laying open to thepublic of any U.S. or foreign patent application, whichever comes first,and for the availability of these seeds to one determined by the U.S.Commissioner of Patents and Trademarks (or by any counterpart to theCommissioner in any patent office in any other country) to be entitledthereto under pertinent statutes and regulations. The assignee of thepresent application has agreed that if any of the seeds on depositshould become nonviable or be lost or destroyed when cultivated undersuitable conditions, they will be promptly replaced on notification witha viable sample of the same seeds.

Five other lines, designated by the inventor as PWC17, PWC19, PWC21,PWC22, and CMC27, exhibited lower levels of herbicide resistance. Theselines appear to differ both from the lines that have now been depositedwith ATCC, and from prior line ATCC 97523. Due to their lower levels ofresistance, these lines had not been deposited with ATCC as of the 10May 2000 priority date of the present application. However, these linesmay have potential value as breeding material to cross with othersources of herbicide resistance, or with each other, in order to enhancetotal levels of resistance. If these five lines involve differentresistance mechanisms, or different AHAS isozymes as compared to theATCC-deposited lines, then crossing one of these lines with one of theATCC-deposited lines could result in a hybrid with an enhanced totallevel of resistance. Their herbicide resistance levels would not,however, appear to make any of these five lines, standing alone,suitable candidates for breeding new herbicide resistant rice lines.

EXAMPLE 28

Mutations were induced in seeds of ten rice varieties by exposure eitherto gamma rays or to EMS. Ten-pound lots of seed of each variety weresubjected to 25 k-rad of gamma irradiation from a Cobalt-60 source atthe Nuclear Science Center, Louisiana State University, Baton Rouge, La.prior to planting. An additional ten pounds of seed of each variety wasdivided into three equal portions; and each portion was soaked for 16hours in either 0.1%, 0.5%, or 1% EMS immediately prior to planting.Several hundred pounds of seed were harvested from plants grown from theseeds subjected to these mutagenic treatments.

The following spring the harvested seed was planted in strips in a fieldplanting occupying a total of about three acres. At the 3-4 leaf stageof seedling development, herbicides were applied to screen forherbicide-resistant mutants. Half of the seedlings of each variety weresprayed with a 2× treatment of nicosulfuron, and half were sprayed witha 2× treatment of imazethapyr, in both cases by a tractor-mountedsprayer. Nicosulfuron was applied at the rate of 0.063 lb activeingredient (a.i.) per acre, and imazethapyr was applied at 0.125 lb a.i.per acre. Non-ionic surfactant (0.25%) was added to each spray solution.Approximately 35 million rice seedlings were sprayed in this manner.About four weeks later a single surviving plant was identified. Thesurviving plant was in a strip that had been sprayed with imazethapyr,and was derived from the “parent” rice variety “AS3510,” treated byexposure to 0.5% EMS. No symptoms of injury from the herbicide treatmentwere evident on this plant at the time it was discovered, while all theother plants were either severely injured or dead. The plant wastransferred to the greenhouse for seed increase and further testing.

Subsequent testing in the greenhouse and field demonstrated that theprogeny of this rice plant possessed resistance to severalAHAS-inhibiting herbicides, including at least the following herbicides:imazethapyr, nicosulfuron, imazaquin, imazameth, imazapyr, and imazamox.AHAS enzyme assays indicated that this rice line possessed a mutant AHASenzyme that was responsible for resistance to AHAS-inhibitingherbicides.

A sample of the seed from this rice line, designated 93AS3510 or SPCW-1,was deposited with the American Type Culture Collection (ATCC), currentaddress 10801 University Boulevard, Manassas, Va. 20110-2209 on 25 Apr.1996, and was assigned ATCC Accession No. 97523. This deposit was madepursuant to a contract between ATCC and the assignee of this patentapplication, Board of Supervisors of Louisiana State University andAgricultural and Mechanical College. The contract with ATCC providesthat these seeds or the progeny of these seeds are now availablepermanently and without restriction to the public. The assignee of thepresent application has agreed that if any of the seeds on depositshould become nonviable or be lost or destroyed when cultivated undersuitable conditions, they will be promptly replaced on notification witha viable sample of the same seeds.

Further Field Tests and Greenhouse Tests

Further field tests and greenhouse tests were conducted to evaluate thetolerance of the resistant lines. The field tests included bothpre-emergence and post-emergence herbicide application studies. The samelines were included in both studies, except that line WDC37 was includedin the pre-emergence study only, due to the lack of sufficient quantityof seed at the time of the study.

The herbicides applied as pre-emergence applications were imazaquin,imazethapyr, and imazapic. Each treatment was applied to each of tworeplicate plots. Each replicate plot contained three-foot long rows ofeach herbicide resistant line, along with a check row of non-resistantrice. Two plots were left unsprayed to serve as untreated controls. Allherbicide-resistant lines exhibited little or no injury from theherbicide applications. All check rows of the non-resistant rice varietyCypress, by contrast, were either killed or severely injured in allplots given herbicide treatments.

Post-emergence application was studied in fifty replicate plots of thesame herbicide-resistant lines, except that line WDC37 was not includedin the post-emergence field study. For the post-emergence field study,each herbicide treatment was applied to each of two replicate plots.Four plots were left unsprayed to serve as untreated controls. Herbicidetreatments studied post-emergence were imazethapyr (Pursuit™ orNewpath™), imazapic (Cadre™), imazamox (Raptor™), a 1:1 (by weight)mixture of imazapic and imazapyr, a 3:1 (by weight) mixture of imazapic(Cadre™) and imazapyr (Arsenal™), imazapyr (Arsenal™), chlorimuron ethyl(Classic™), metsulfuron methyl (Ally™), nicosulfuron (Accent™),rimsulfuron (Matrix™), a 2:1 mixture (by weight) (Harmony Extra™) ofthifensulfuron methyl and tribenuron methyl, and pyrithiobac sodium(Staple™).

The greenhouse tests comprised two replicate studies using the sameherbicides and rates as were used in the post-emergence field test. Thegreenhouse studies evaluated the post-emergence herbicide resistance ofa few lines for which the quantity of seed then available was inadequateto include in the field tests. Seeds of the resistant lines were plantedin 2 inch×2 inch peat pots, and the seedlings were then sprayed at the3-4 leaf stage. Non-resistant check lines were included for comparison.As in the field tests, the non-resistant checks were either killed orseverely injured by the herbicide treatments.

The results of these field and greenhouse studies are summarized inTables 3 and 4.

Results and Discussion

Previous selections for imidazolinone-resistant rice by screeningfollowing seed exposure to EMS had resulted in fewer resistant ricelines. For example, screening approximately 35 million M₂ seed followingexposure of the M₁ seed to 0.1%, 0.5%, or 1.0% EMS for 16 hours resultedin a single herbicide-resistant mutant plant (ATCC 97523), for a successratio of 1 resistant mutant per 35 million mutated seed. By contrast,each of the two later series of screenings had a significantly higherrate of successfully producing herbicide-resistant mutants. It isbelieved, without wishing to be bound by this theory, that the improvedefficiency was due to the difference in mutagen concentrations andexposure times used.

The more efficient mutation protocols described here used a relativelylonger exposure to a relatively lower concentration of mutagen than hadpreviously been used. In Examples 1-15 the average mutagen exposure timewas 22.75 hours, and the EMS concentration was 0.175%. This represents a42% longer average exposure time, and a 65% reduction in the mutagenconcentration, as compared to the only successful event from the earlierscreening of 35 million seeds (Example 28). The result was a ten-foldincrease in the rate of resistant mutant recovery (one per 3.5 millionseed versus one per 35 million seed).

Examples 16-27 used conditions similar to those for Examples 1-15, andwere also more efficient in producing resistant mutants. The same EMSmutagen concentration (0.175%) was used, and only a slightly differentexposure time (23 hours versus an average of 22.75 hours). Theherbicide-resistant mutant production rate in this trial was 1 plant per5 million seed. These results indicate that longer exposures to lowermutagen concentrations appear generally to produce higher rates ofsuccessful herbicide resistant mutants.

Each of the resistant mutants from these two screenings exhibitedresistance to one or more imidazolinone and sulfonylurea herbicides. Asummary of the herbicide applications used in the initial screening forresistance is given in Table 1. The results of the field tests forExamples 1-15 (SSC01 through SSC15) are given in Table 2. The results ofthe field tests for Examples 16-27 (those resistant lines having PWC,CMC, or WDC designations) are given in Tables 3 and 4. Note that theapplication rates in Tables 1, 2, and 3 are given in pounds of activeingredient per acre, while the rates in Table 4 are given in ounces ofactive ingredient per acre.

TABLE 1 Screening Herbicide Application Screening Herbicide Application(lb ai/A) Imazethapyr 0.125 pre-emerge + Imazapyr Imazameth Line 0.063post-emerge 0.10 post-emerge 0.125 post-emerge SSC01 X SSC02 X SSC03 XSSC04 X SSC05 X SSC06 X SSC07 X SSC08 X SSC09 X SSC10 X SSC11 X SSC12 XSSC13 X SSC14 X SSC15 X PWC16 X PWC17 X PWC18 X PWC19 X PWC20 X PWC21 XPWC22 X PWC23 X PWC24 X CMC25 X CMC26 X CMC27 X CMC28 X CMC29 X CMC30 XCMC31 X WDC32 X WDC33 X WDC34 X WDC35 X WDC36 X WDC37 X WDC38 X

TABLE 2 Post-Screening Herbicide Testing Herbicide Application Rate (lbai/A); & whether applied pre-emergence or post-emergence Imazethapyr(75%) + Imazapyr Sulfometuron Imazethapyr Imazapyr Imazameth (25%)Methyl Nicosulfuron 0.125 0.063 0.125 0.188 0.05 0.09 0.063 0.125 0.0520.10 0.05 0.10 0.05 Line pre post post post post post post post post prepost pre post SSC01 X X X X X X X X X 0 0 X X SSC02 X X X X X X X X X 00 X X SSC03 X X X X X X X X X 0 0 X X SSC04 X X X X X X X X X X 0 X XSSC05 X X X X X X X X X 0 0 X X SSC06 X X X X X X X X X X 0 X X SSC07 XX X X X X X X X 0 0 X X SSC08 X X X X X X X X X X 0 X X SSC09 X X X X XX X X X 0 0 X X SSC10 X X X X X X X X X X 0 X X SSC11 X X X X X X X X X0 0 X X SSC12 X X X X X X X X X 0 0 X X SSC13 X X X X X X X X X 0 0 X XSSC14 X X X X X X X X X 0 0 X X SSC15 X X X X X X X X X 0 0 X X PWC16 XX 0 0 X X PWC17 X X X 0 X X PWC18 X X 0 0 X X PWC19 X X 0 0 X X PWC20 XX 0 0 X X PWC21 X X 0 0 X X PWC22 X PWC23 X X X X X X PWC24 X X X 0 X XCMC25 X X X 0 X X CMC26 X X 0 0 X X CMC27 CMC28 X X 0 0 X X CMC29 X X 00 X X CMC30 X X 0 0 X X CMC31 X X 0 0 X X WDC32 X X X X X X WDC33 X X 00 X X WDC34 X X X 0 X X WDC35 X X 0 0 X X WDC36 X X 0 0 X X WDC37 X X 00 X X WDC38 X X 0 0 X X Notes to Table 2: X = resistant; 0 = sensitive(exhibited wild-type reaction to herbicide); blank = not yet tested.

TABLE 3 Post-Screening Herbicide Testing Herbicide Application Rate (lbai/A); & whether applied pre-emergence or post-emergence ImazethapyrImazapic Imazaquin Imazamox Imazapic 0.05 + 0.063 0.125 0.188 0.0630.125 0.037 0.075 0.15 0.075 0.15 0.125 0.25 0.375 0.05 0.10 Imazapyr0.05 Line pre pre pre post post pre pre pre post post pre pre pre postpost post SSC01 X X X X X X X X X X X X X X X X SSC02 X X X X X X X X XX X X X X X X SSC03 X X X X X X X X X X X X X X X X SSC04 X X X X X X XX X X X X X X X X SSC05 X X X X X X X X X X X X X X X X SSC06 X X X X XX X X X X X X X X X X SSC07 X X X X X X X X X X X X X X 0 X SSC08 X X XX X X X X X X X X X X X X SSC09 X X X X X X X X X X X X X X X X SSC10 XX X X X X X X X X X X X X X X SSC11 X X X X X X X X X X X X X X X XSSC12 X X X X X X X X X X X X X X X X SSC13 X X X X X X X X X X X X X X0 X SSC14 X X X X X X X X X X X X X X 0 X SSC15 X X X X X X X X X X X XX X 0 X PWC16 X X X X X X X X X X X X X X X X PWC23 X X X X X X X X X XX X X X X X CMC29 X X X X X X X X X X X X X X X X CMC31 X X X X X X X XX X X X X X X X WDC33 X X X X X X X WDC37 X X X X X X X X X WDC38 X X XX X X X X X X X X X X X X Herbicide Application Rate (lb ai/A); &whether applied pre-emergence or post-emergence Imazapic 0.075 +Imazapic 0.15 + Imazapyr 0.025 Imazapyr 0.05 Imazapyr Line post post0.05 post 0.10 post SSC01 X X X X SSC02 X X X X SSC03 X X X X SSC04 X XX X SSC05 X X X X SSC06 X X X X SSC07 X X X X SSC08 X X X X SSC09 X X XX SSC10 X X X X SSC11 X X X X SSC12 X X X X SSC13 X X X X SSC14 X X X XSSC15 X X X X PWC16 X X X X PWC23 X X X X CMC29 X X X X CMC31 X X X XWDC33 X X X X WDC37 WDC38 X X X X Notes to Table 3: X = resistant; 0 =sensitive (exhibited wild-type reaction to herbicide); blank = not yettested.

TABLE 4 Post-Screening Herbicide Testing Herbicide Application Rate(ounces ai/A); & whether applied pre-emergence or post-emergenceThifensulfuron methyl (66.7%) + Chlorimuron Metsulfuron tribenuronmethyl Pyrithiobac Ethyl Methyl Nicosulfuron Rimsulfuron (33.3%) sodium0.125 0.250 0.06 0.12 0.5 1.0 0.20 0.40 0.45 0.90 1.0 2.0 Line post postpost post post post post post post post post post SSC01 X X X X X X X XX X X X SSC02 X X X X X X X X X X X X SSC03 X X X X X X X X X X X XSSC04 X X X X X X X X X X X X SSC05 X X X X X X X X X X X X SSC06 X X XX X X X X X X X X SSC07 X X X X X X X X X X X X SSC08 X X X X X X X X XX X X SSC09 X X X X X X X X X X X X SSC10 X X X X X X X X X X X X SSC11X X X X X X X X X X X X SSC12 X X X X X X X X X X X X SSC13 X X X X X XX X X X X X SSC14 X X X X X X X X X X X X SSC15 X X X X X X X X X X X XPWC16 0 0 X X X 0 0 0 X X X X PWC23 0 0 X X X 0 0 0 X X X X CMC29 0 0 0X X 0 0 0 X X X X CMC31 0 0 0 X X X 0 0 X X X X WDC33 X X X X 0 0 0 0 XX X X WDC37 WDC38 0 0 0 X X X 0 0 0 X X X Notes to Table 4: X =resistant; 0 = sensitive (exhibited wild-type reaction to herbicide);blank = not yet tested. In the entries for CMC29, CMC31, and WDC38 formetsulfuron methyl, and also for WDC38 (only) for the thifensulfuronmethyl-tribenuron methyl mixture, at the lower rate of application, theresponse was identical to that of the wild-type, with all surviving thelower rate of application; while at the higher rate of application, thewild-type plants were seriously injured, and the CMC29, CMC31, and WDC38lines exhibited substantially less injury.

Further examination of these plants led to the conclusion that thefollowing herbicide resistant lines appeared to be identical to priorherbicide resistant line ATCC 97523, presumably because a few seeds ofATCC 97523 from prior trials had remained dormant in the soil betweengrowing seasons: PWC18, PWC20, PWC24, CMC25, CMC26, CMC28, CMC30, WDC32,WDC34, WDC35, and WDC36.

Germination studies have confirmed the high levels of pre-emergenceresistance displayed by several of the novel rice lines. Typically, whengermination of a non-resistant line occurs in concentrations ofimidazolinone in solution as low as 1-2 ppm, significant injury or deathoccurs. For the novel lines PWC16, PWC23, CMC29, CMC31, WDC33, WDC37,and WDC38, as well as for the parental (non-resistant) Cypress line,germination studies were conducted as follows. For each line, 15 seedsper dish in three replicate Petri dishes were germinated atconcentrations of 0, 10, 20, 30, 40, 50, and 60 ppm for each herbicidetested. The parental Cypress line failed to germinate at concentrationsof 10 ppm and higher for each of the herbicides tested. By contrast,each of the experimental lines germinated, to varying degrees (somebetter than others) at herbicide concentrations of 60 ppm, except asnoted. With imazapyr, all experimental lines germinated to some extentat 60 ppm. With imazethapyr, all experimental lines germinated to someextent at 60 ppm. With imazamox, all experimental lines germinated tosome extent at 60 ppm, except WDC38. With imazapic, only PWC16 and PWC23germinated at 60 ppm. Thus the novel lines germinated in concentrationsof herbicide 60 times that which ordinarily kills or causes significantinjury to wild-type rice plants.

Enhanced resistance will result from crossing the novel rice lines withone another. Enhanced resistance will also result from the synergy ofcrossing one or more of the novel rice lines, with their resistant AHASenzymes, with the metabolic-based resistant rice lines disclosed in U.S.Pat. No. 5,545,822, as typified by the rice having ATCC accession number75295. As disclosed in the present inventor's published internationalpatent application WO 97/41218, such synergy has been seen in hybrids ofthe rice having ATCC accession number 75295 with the rice having ATCCaccession number 97523, the latter having a mutant, resistant AHASenzyme in rice.

Notes on Mutation Selection Procedures in the Field

The following procedures were used for screening large quantities ofmutated rice seed for herbicide resistance in the field.

Exposure to mutagen or to conditions conducive to the induction ofmutations may be performed at different stages of growth and differentculture conditions, e.g., exposing to mutagen dry seed, seed sprouted inwater for 24 hours, or seed sprouted in water for 48 hours, etc.; orgrowing cells in tissue culture, such as another culture, with orwithout the contemporaneous application of mutagen; and the like.

Rice to be planted for seed is ordinarily cleaned after harvest. Oncecleaning is completed, any standard planting equipment can besatisfactorily used. However, this laborious and time-consuming cleaningstep can be bypassed if the planting equipment will tolerate the piecesof straw and other extraneous material that typically accompanycombine-harvested rice. Eliminating the cleaning step allows generationsof seed to be grown, screened, and increased more rapidly. For example,using a spinner/spreader attachment on a tractor allows broadcastplanting of rice that is accompanied by a moderate amount of extraneousmaterial. Broadcast planting is also more rapid than drill-seeding,saving further time and labor. Seed planted with a spinner/spreader caneither be lightly incorporated into the soil followingbroadcast-spreading, or allowed to remain on the soil surface, in whichcase it must be kept sufficiently moist by irrigation if rainfall isinadequate.

Freshly-harvested rice seed may have a degree of dormancy, whichprevents some of the otherwise viable seed from sprouting immediately.This dormancy normally disappears during storage. However, if theharvested seed is to be planted for selection purposes shortly afterharvest to accelerate generation time, then treatment to reduce oreliminate dormancy is beneficial. One method to eliminate dormancy is toexpose the seed to a temperature of about 50° C. for about five days;but temperatures significantly higher may injure the rice seeds.Moisture should be allowed to escape from the seed during thistreatment, so relatively small containers of moisture-permeable materialshould be used, such as cloth bags. Alternatively, stems with paniclesstill attached may be positioned to allow air to circulate over thepanicles, for example, by standing them upright in a paper bag. As afurther alternative, forced-air drying may be used, with or withoutstorage in bags, provided that the seed is situated so that moisture isnot entrapped around portions of the grain.

When spraying mutated rice seed or plants to identify resistantindividuals, it is important to achieve as uniform and precise atreatment as possible. Since the number of true resistant individualswill be a very small fraction of the total number of seeds, even a smallfraction of “escapes” (i.e., false positives, plants fortuitously notreceiving any herbicide) can complicate and retard the screeningprocess. Therefore the herbicide-spraying equipment should be in goodcondition, and should be calibrated as accurately as possible. Eachspray nozzle along the spraying boom should deliver spray at the samevolumetric rate. Nozzles should be accurately aligned to avoidinsufficient spray overlap between nozzles. Relatively short tractorspray booms (for example, approximately 12 feet) are helpful inminimizing undesirable boom movements while spraying.

Appropriate nozzles include the following, each of which has a flatspray tip, and sprays approximately 15 gallons per acre at 40 pounds persquare inch (gauge) spray pressure, with a 20-inch nozzle spacing, atthe indicated ground speeds: 8001VS (2 mph), 80015VS (3 mph), 8002VS (4mph), 8003VS (5 mph). (Spraying Systems Co., Wheaton, Ill.) To optimizethe spray pattern, the nozzle height above the target (either the top ofthe plant canopy or the soil) should be adjusted so that the spraypattern from each nozzle overlaps the spray pattern from each adjacentspray nozzle by about 30% (as measured linearly). Using the 80 degreenozzles listed above, at a 40 psi spraying pressure, and a 20 inchspacing between nozzles, an optimum spray height above the target wouldbe 17 to 19 inches. Holding other parameters constant, but changing thenozzle spacing to 30 inches, an optimum spray height would increase to26 to 28 inches. Using spray pressures lower than 40 psi will typicallyreduce the nozzle spray angles, and adjusting to a lower spray heightmay be necessary to achieve proper overlap at lower pressures. All sprayequipment should be precisely calibrated before use.

When spraying, carefully measured marking flags to guide the spray-rigoperator are frequently beneficial, as are flags at midfield in largerfields, in addition to those at the ends of the fields. Wind speedshould be essentially zero, a condition that is often seen in the earlymorning or late afternoon. Spraying should not be performed if rain isanticipated within about the next six hours (a time that varies,depending on the particular herbicide). Pre-emergence spraying should beapplied to dry ground. If the herbicide requires moisture foractivation, then irrigation or rainfall after planting is required.

Uniformity of spraying is best accomplished by dividing the herbicide tobe applied equally between two consecutive sprayings, one after theother. The spray solution is prepared at half the final treatmentconcentration. Two passes are then made in opposite directions toachieve the desired total treatment concentration. For example, if thefirst pass on a particular row is made in the North-to-South direction,the second pass is made in the South-to-North direction. When sprayingwith a tractor, this may be accomplished by traveling in the oppositedirection in the same tracks for the second application.

Complete coverage is promoted by using large spray volumes (i.e., diluteconcentrations of herbicide) and small spray droplet size. Spray volumesof 30 to 40 gallons per acre have worked well, particularly with twoapplications of 15 to 20 gallons per acre each. Spray pressures of 30 to40 pounds per square inch (gauge) have worked well in producing finesprays that provide thorough coverage. Nozzles should be evenly spaced,preferably about 20 inches apart.

The total rate of herbicide application used for the selection ispreferably at least twice the normal use rate for the same herbicide.For example, if 0.063 lb ai/A is the normal use rate for crops, then anappropriate concentration to select for resistant individuals would betwo applications at the same rate, resulting in 0.125 lb ai/A totaltreatment.

The combination of two sprayings, large spray volumes, high spraypressures, and an elevated treatment concentration helps minimize theoccurrence of escapes, i.e., individuals that are not truly resistant,but that survived the procedure simply because they were inadequatelysprayed.

There are advantages to conducting selection with herbicides thatpossess both soil and foliar activities. The soil activity of theherbicide can be used directly to select for resistant individuals thatgrow despite the pre-emergence application. Alternatively, apre-emergence application can be used to eliminate a large percentage ofthe non-resistant entities, following which a foliar application is madeon the surviving individuals. This early thinning of the stand densitygreatly reduces the problem of spray interception that can otherwiseoccur within a thick stand of young seedlings, i.e., the possibility ofa seedling that is physically shielded from the spray by otherseedlings.

Using both soil and foliar application of a suitable herbicide alsoreduces the problem of “escapes,” because the herbicide's soil activitywill often eliminate individuals that might otherwise escape the foliarspray. When using a herbicide having primarily, or only, foliaractivity, an additional spraying may be necessary for two reasons. Onereason is to eliminate non-resistant individuals that escaped the foliarspray. Also important is the elimination of non-resistant individualsfrom late-sprouting seed. A plant that grows from a seed that sproutsafter spraying will not be controlled by a herbicide having only foliaractivity. Within two weeks, such a plant may reach a size that makes itappear to be a resistant mutant that survived the foliar treatment. If asecond foliar spraying either is undesirable or is not feasible, analternative is to leave a small area of the field unsprayed whenapplying the first application, to provide a direct standard fordetermining the size that resistant seedlings should achieve during theintervening period.

Using a herbicide with both soil and foliar activity also presents theopportunity to select efficiently for both pre-emergence andpost-emergence resistance within the same individual plants. Thisselection is accomplished by applying sequential applications. Ifperformed properly, the likelihood will then be high that individualssurviving sequential applications are resistant to both pre- andpost-emergence treatments with that herbicide, rather than escapes.

As the selection procedure is in progress, care should be taken that thefew surviving individuals are not eaten by birds or insects. Avoidingsuch predation is important for both post-emergence and pre-emergencetreatments. Sound-making devices may be used to drive away birds, suchas blackbirds, that consume rice seeds and small seedlings. Insects suchas fall armyworms and rice water weevils also may kill small survivors,and the application of an insecticide on a preventative basis isfrequently desirable. Daily monitoring of the situation should beundertaken if an investigator chooses not to use bird-discouragingdevices or insecticides preventatively.

Assays for Total AHAS Activity

The procedures used to assay the activity of the acetohydroxyacidsynthases from various rice lines as reported below were substantiallyas described in B. K. Singh et al., “Assay of AcetohydroxyacidSynthase,” Analytical Biochemistry, vol. 171, pp. 173-179 (1988), exceptas noted. In the first paragraph of Singh's “Materials and Methods,”instead of corn suspension culture cells, shoot tissues fromgreenhouse-grown rice seedlings at the 3-4 leaf stage of development, orrice suspension culture cells were used. For shoot tissues, 40.0 or 50.0grams (fresh weight) of tissue were extracted in the same manner foreach of the breeding lines; for Cypress suspension cells, 16.0 grams ofcells were used, harvested eight days after subculture. At thesuggestion of the first author, B. K. Singh (personal communication),the desalting step mentioned at the bottom of Singh's first column under“Materials and Methods” was eliminated. Pursuit™ herbicide (imazethapyr,also known as Newpath™) or Arsenal™ herbicide (imazapyr) was included inthe “standard reaction mixture” for the AHAS assay in variousconcentrations. Colorimetric absorbance was measured at 520 nm. Checkswere made of direct acetoin formation during the enzyme assay.

The following nine rice lines have been assayed in this manner to date:the non-resistant Cypress line (the parental line for some of theherbicide resistant lines), ATCC 97523, PTA-904, PTA-905, PTA-902,PTA-903, PTA-906, PTA-907, and PTA-908. Some assays were conducted atdifferent times, and assays at some herbicide concentrations wererepeated. Differences were noted among the lines with respect to totalAHAS enzyme activity and the levels of herbicide resistance. In themodified Singh assay for total AHAS activity, using crude enzymeextract, in the absence of herbicide, most (but not all) of theherbicide-resistant lines expressed greater total AHAS activity than didthe non-resistant Cypress line. Following treatment with the herbicidesPursuit™ (imazethapyr, also known as Newpath™) or Arsenal™ (imazapyr),the reduction in AHAS activity was greater in the Cypress line than inany of the resistant lines assayed. For the line that has appeared tohave the highest resistance in testing to date, PWC23 (PTA-905), enzymeactivity in the presence of very high herbicide levels (1000 μM ofeither imazethapyr or imazapyr) was similar to the enzyme activity ofthe nonresistant Cypress line in the absence of any herbicide. All theresistant lines assayed expressed resistance to both imazethapyr andimazapyr, while the nonresistant Cypress line was sensitive to bothherbicides. Results are shown in Table 5. In Table 5, the first row (“Noherbicide”) is reported as absorbance at 520 nm. All other entries in agiven column (i.e., for a given line of rice) are reported as apercentage of the absorbance for the same rice line in the absence ofherbicide.

TABLE 5 Total AHAS Activity, Crude Enzyme Extracts, measured asabsorbance at 520 nm ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC 97523PTA-904 PTA-905 PTA-902 PTA-903 PTA-906 PTA-907 PTA-908 Cypress(93AS3510) (PWC16) (PWC23) (CMC29) (CMC31) (WDC33) (WDC37) (WDC38) Noherbicide 0.766 0.837 0.713 1.107 0.811 1.038 0.851 1.226 0.822 50 μM63% 101%  95% 99% 92% 84% 89% 92% 95% imazethapyr 100 μM 54% 92% 83% 88%88% 82% 85% 86% 90% imazethapyr (first replicate) 100 μM 58% 91% 92% 93%93% 87% 86% — — imazethapyr (second replicate) 1000 μM 56% 78% 78% 80%84% 81% 82% 64% 66% imazethapyr 50 μM 63% 91% 90% 95% 92% 86% 88% 84%81% imazapyr 100 μM 57% 83% 83% 88% 84% 80% 74% 78% 78% imazapyr 1000 μM45% 68% 76% 75% 75% 68% 73% 66% 66% imazapyr

The results shown in Table 5 clearly show that each of the resistantlines listed in that Table (ATCC 97523, ATCC PTA-904, etc.) contains aresistant mutant AHAS enzyme. The lowest concentrations tested, 50 μM ofimazethapyr or imazapyr, reduced the activity of the non-resistantline's AHAS to about 63% of control—a reduction in activity that is morethan ample to be lethal to plants in the field. By contrast, theresistant lines had AHAS activities ranging from 84% to 101% of controlat these herbicide concentrations. Even at the highest herbicideconcentrations tested, 1000 μM, enzyme activities in the resistantplants ranged from 64% to 84%, versus 45% or 56% for the non-resistantline. Put differently, each of the resistant plants showed higher AHASactivity at the extremely high herbicide concentration of 1000 μM thanthe AHAS activity of the non-resistant line at the lowest herbicide ratetested, 50 μM. In fact, the absolute activity exhibited by the mostresistant line, PTA-905, at the highest 1000 μM herbicide rates tested(activities of 0.883 for imazethapyr and 0.834 for imazapyr) were higherthan the AHAS activity for the non-resistant Cypress line in the absenceof any herbicide (0.766).

The results given in Table 5 therefore clearly demonstrate that theherbicide resistance characteristics of at least the resistant ricelines listed in Table 1 were due to a resistant mutant AHAS enzyme.

Germination Inhibition Levels

Pre-emergence herbicide applications were tested to identify the levelsof two different herbicides that would completely inhibit germinationfor several rice lines. Seed of each line tested was germinated in aplastic disposable petri dish containing 8 mL of herbicide solution anda layer of Whatman No. 4 filter paper. The fungicide Vitavax 200 at aconcentration of 0.5 mL/L was added to the incubation solutions toinhibit fungal growth. Untreated controls were incubated in solutionscontaining fungicide but no herbicide. Twenty seeds were placed in eachof 3 replicate dishes per treatment, and were incubated at 25° C. under16 hour: 8 hour light/dark photoperiods at a fluorescent light intensityof 15 micro-Einsteins per square meter per second. (One Einstein=1 moleof photons.) Treatments were evaluated 11 days after incubation. Theresults of these pre-emergence experiments are shown in the Table 6,which indicates the herbicide concentrations, in parts per million,needed to completely inhibit the germination of the lines tested.

TABLE 6 Herbicide Concentrations (ppm) needed to completely inhibitgermination. Imazapic Imazethapyr (Pursuit ™ (Cadre ™) or Newpath ™)Cypress 0.5 1 ATCC 97523 10 10 (93AS3510) ATCC PTA-904 60 80 (PWC16)ATCC PTA-905 90 100 (PWC23) ATCC PTA-902 50 90 (CMC29) ATCC PTA-903 7060 (CMC31) ATCC PTA-906 50 70 (WDC33) ATCC PTA-907 50 60 (WDC37) ATCCPTA-908 30 30 (WDC38)

As shown Table 6, each of the lines ATCC PTA-904, ATCC PTA-905, ATCCPTA-902, ATCC PTA-903, ATCC PTA-906, ATCC PTA-907, and ATCC PTA-908exhibited substantially higher resistance to pre-emergence applicationsof the herbicides imazapic and imazethapyr than did ATCC 97523-higher bya factor of 3 to 10. Also note that the pre-emergence resistancecharacteristics of each of these seven lines to imazapic and imazethapyrclearly demonstrate that they are different from the line ATCC 97523;similarly, see also the results reported in Tables 7 and 8 below.

Further Greenhouse and Field Tests

The post-emergence resistance of the new lines has also been tested: inpeat pots in a greenhouse, and on plants in the field. In the peat pottest, the tolerance of fourth generation (M₄) plants to variouspost-emergence imidazolinone herbicide treatments was tested. Individualseedlings, in 3 replicate peat pots per treatment, were sprayed at the2-3 leaf stage with 0×, 5×, 10×, 15×, and 20× herbicide treatments.Plants were rated 42 days after treatment. The values listed in Table 7below were the highest tested rates that were tolerated with no visibleinjury, in some cases accompanied by a value in parentheses giving ahigher rate at which the plants survived, but with injury.

TABLE 7 Highest Rates of Post-Emergence Herbicide Treatment Toleratedwithout visible injury ATCC ATCC ATCC ATCC ATCC ATCC ATCC ATCC 97523PTA-904 PTA-905 PTA-902 PTA-903 PTA-906 PTA-907 PTA-908 Cypress(93AS3510) (PWC16) (PWC23) (CMC29) (CMC31) (WDC33) (WDC37) (WDC38)Imazethapyr All died at All died at 10X 15X 15X 10X 15X 10X All died at(Pursuit)  5X  5X (survived (survived (survived (survived (survived(survived  5X  15X)  20X)  20X)  15X)  20X)  15X) Imazamox All died atAll died at All died at  5X injured at  5X  5X  5X 10X (Raptor)  5X  5X 5X (survived  5X (survived  10X)  10X) Imazapyr All died at All died at10X 10X 10X 10X 10X 10X 10X (Arsenal)  5X  5X Imazapic All died at Alldied at  5X 10X 10X All died at  5X  5X injured at (Cadre)  5X  5X  5X(survived  5X  10X) Notes: (1) Except for imazamox, in each case a 10Xapplication = 0.63 lb ai/A = 706 g ai/ha, and all other rates ofapplication are proportional. For imazamox, a 10X application = 0.32 lbai/A = 359 g ai/ha. (2) Because the number of replicates in thisparticular experiment was low, and because the lines were stillsegregating, a higher degree of confidence may be aproppriate for thepositive results in Table 7 (herbicide tolerance) than for the negativeresults (herbicide susceptibility).

Field tests were conducted to evaluate the herbicide-resistance of linesPTA-902, PTA-903, PTA-904, PTA-905, and PTA-908. The same tests wereconducted on both the non-resistant rice variety Cypress, and theherbicide resistant rice line ATCC 97523. All lines were planted in1-meter rows, with two replications of each treatment. Post-emergencetreatments of various herbicides were applied when the rice was at the2-3 leaf stage of development. Herbicide applications were made with abackpack sprayer at a spray rate of 15 gallons per acre (163 liters perhectare). Evaluations of herbicide resistance were made as the plantsreached the flowering stage, and were based on relative performance ascompared to the non-treated control rows of the same lines. Technicaldifficulties in conducting this set of experiments prevented theacquisition of good data for line PTA-907. Line PTA-906 was evaluated ingreenhouse tests, rather than field tests, in order to conserve thelimited amount of seed that was available for this line at the time.Individual plants in 5 cm by 5 cm peat pots were sprayed at the 2-3 leafstage with the same herbicide applications as were used in thepost-emergence field tests. Herbicide treatments were also made with abackpack sprayer at a spray rate of 15 gallons per acre (163 liters perhectare). Evaluations of herbicide resistance were made as the plantsreached the flowering stage, and were based on relative performance ascompared to the non-treated controls of the same lines in thegreenhouse. The greenhouse evaluation was conducted twice, and eachtreatment was replicated. As in the field test, controls were conductedfor comparison with non-resistant plants (in this case, thenon-resistant Bengal variety was used) and with the earlier ATCC 97523plants. Results are shown in Table 8. (Several additional herbicidetreatments, not shown in Table 8, were also conducted.)

TABLE 8 Post-Emergence Herbicide Tolerance in Field Trials or GreenhouseTrials. Entries give percent injury as compared with untreated controlsof the same lines. Imazapic (Cadre) 0.075 lb Rimsulfuron ai/A + imazapyr(Arsenal) (Matrix) 0.025 lb ai/A (=84 and 28 g 0.025 lb ai/A ai/ha,respectively) (=28 g ai/ha) Cypress (field) 100% 100% ATCC 97523 95% 90%(93AS3510) (field) ATCC 203419 28% 18% (SSC01) (field) ATCC 203420 60%20% (SSC02) (field) ATCC 203421 38% 18% (SSC03) (field) ATCC 203422 23%18% (SSC04) (field) ATCC 203423 75% 20% (SSC05) (field) ATCC 203424 8%8% (SSC06) (field) ATCC 203425 23% 75% (SSC07) (field) ATCC 203426 15%18% (SSC08) (field) ATCC 203427 8% 13% (SSC09) (field) ATCC 203428 8%18% (SSC10) (field) ATCC 203429 48% 45% (SSC11) (field) ATCC 203430 33%18% (SSC12) (field) ATCC 203431 30% 20% (SSC13) (field) ATCC 203432 63%33% (SSC14) (field) ATCC 203433 43% 35% (SSC15) (field) ATCC PTA-904 0%100% (PWC16) (field) ATCC PTA-905 3% 100% (PWC23) (field) ATCC PTA-9020% 100% (CMC29) (field) ATCC PTA-903 0% 100% (CMC31) (field) ATCCPTA-908 5% 100% (WDC38) (field) Bengal (greenhouse) 100% 100% ATCC 97523100% 60% (93AS3510) (greenhouse) ATCC PTA-906 3% 100% (WDC33)(greenhouse) ATCC PTA-907 N/A N/A (WDC37)

Sequencing, Cloning, and Plant Transformation

Repeated attempts by the inventor and his colleagues to sequence therice AHAS coding sequence by PCR amplification of genomic rice DNA overan extended period of time had not been successful. These difficultieshave now been overcome by RT-PCR amplification of RNA instead.

The resistant mutant AHAS nucleotide sequences from the rice plantshaving accession numbers ATCC 97523, 203419, 203420, 203421, 203422,203423, 203424, 203425, 203426, 203427, 203428, 203429, 203430, 203431,203432, 203433, PTA-904, PTA-905, PTA-902, PTA-903, PTA-906, PTA-907,and PTA-908 are sequenced and cloned, along with the wild-type AHASnucleotide sequence from the parental rice varieties AS3510 and Cypress.As of the filing date of the present application, substantial portionsof this work have been completed, as described below. The remainingsequencing and cloning will hereafter proceed to completion usingtechniques known in the art, as described generally below. The sequencesthat have been obtained to date show the nature and locations of some ofthe point mutations responsible for the observed herbicide resistantphenotypes.

The parental non-resistant line Cypress has been publicly released bythe Louisiana State University Agricultural Center's Rice ResearchStation, and is widely available commercially. Samples of the parentalnon-resistant line AS3510 are available upon request without charge fromthe inventor, Timothy P. Croughan, c/o Louisiana State UniversityAgricultural Center, Rice Research Station, P.O. Box 1429, Crowley, La.70527-1429, United States. Samples of line AS3510 may also be availablefrom other rice breeding programs or rice germplasm collections.

EXAMPLE 29 CMC31 (ATCC PTA-903)

Total RNA from the CMC31 line (ATCC accession number PTA-903) wasextracted from rice callus tissue culture with the RNEASY Mini Kit(Qiagen Inc., Valencia, Calif.) using ˜100 mg of tissue. Themanufacturer's “RNeasy Plant Mini Protocol For Isolation of Total RNAfrom Plant Cells and Tissues, and Filamentous Fungi” was followed. TotalRNA was eluted in 50 μl of diethylpyrocarbonate—water solution, for ayield of ˜100 μg.

Next, poly A⁺ RNA (mRNA) was purified from the total RNA with theOligotex mRNA Mini Kit (Qiagen Inc., Valencia, Calif.) following theOligotex mRNA Spin-Column Protocol. Buffer amounts used were thoserecommended by the manufacturer for a Mini Prep size. The mRNA waseluted twice with 25 μL of the OEB buffer from the kit.

Reverse transcriptase-polymerase chain reaction (RT-PCR) results wereobtained using Ready-To-Go RT-PCR Beads (Amersham Pharmacia Biotech,Piscataway, N.J.), following the manufacturer's recommended Two-Stepprotocol for RT-PCR. The PCR primers were based on a published AHASsequence from Hordeum vulgare, GenBank accession no. AF059600. Twoprimer pairs, HvAls-3 & HvAls-4, and HvAls-3 & HvAls-6 (HvAls-3=TGG CGAGGC ACG GCG CCC; HvAls-4=GAC GTG GCC GCT TGT AAG; HvAls-6=AGT ACG AGGTCC TGC CAT) (SEQ ID NOS 6-8, respectively) produced two products, oneapproximately 550 and one approximately 1100 base pairs. (The ˜550 bpproduct was a subset of the ˜1100 bp product, and confirmed the sequenceof the ˜1100 bp product in the region of their overlap.) These productswere electrophoresed on a 1.5% agarose gel at 70 volts for 90 minutes.The bands were excised and purified with the ZymoClean™ Gel DNA RecoveryKit (Zymo Research, Orange, Calif.), and were eluted in 13 μL of 10 mMTris buffer.

The gel-extracted PCR products were sequenced using the Big Dye™ kit (PEApplied Biosystems, Foster City, Calif.), using 5 μL of template per PCRprimer. The resulting 1095 base sequence was analyzed against previouslyreported sequences by the Blast Search software (National Center forBiotechnology Information, available at www.ncbi.nlm.nih.gov/blast/).The sequence is given below as SEQ ID NO 1, in the conventional 5′-3′orientation. These 1095 bases represent about half of the coding portionof the AHAS gene. The inferred partial amino acid sequence is givenbelow as SEQ ID NO 15, in the conventional amino terminus-carboxyterminus orientation.

SEQ ID NO 1 is 85% identical at the nucleotide level (902 bases of 1058)to the AHAS sequence for Zea mays reported as GenBank accession numberX63553; 84% identical (896 bases of 1060 bases) to the AHAS sequence forZea mays reported as GenBank accession number X63554; and 88% identical(603 bases of 678) to the AHAS sequence for Hordeum vulgare reported asGenBank accession number AF059600. It is 97% identical (1062 bases of1088) to the AHAS sequence for Oryza sativa var. Kinmaze reported asaccession number AB049822 (SEQ ID NO 2).

Until the point of the frame shift mutation, SEQ ID NO 15 is 99%identical at the inferred amino acid level (352 amino acids of 355) tothe AHAS sequence for wild type Oryza sativa var. Kinmaze as reported byT. Shimizu et al., “Oryza sativa ALS mRNA for acetolactate synthase,complete cds, herbicide sensitive wild type,” BLAST accession numberAB049822 (April 2001), available through www.ncbi.nlm.nih.gov/blast, SEQID NO 3. At amino acid position 627, there is a Ser-Lys substitution inthe inferred amino acid sequence for line CMC31, followed by asingle-base deletion in the DNA, causing a frame-shift mutation thatgenerally alters the identity of most of the subsequent amino acids(i.e., those between position 627 and the carboxy terminus), and thatintroduces a “stop” seven codons downstream from the codon for aminoacid position 627 (Ser-Lys).

Since the presumptive source of the herbicide resistance mutation hasbeen identified in line CMC31, it is reasonable to infer that thecomplete coding sequence for this line is the same as that of the“parent” wild type Cypress AHAS (SEQ ID NO 14), except for replacing theAGT codon for serine at amino acid position 627 with the substitutionand deletion A-A seen at the corresponding position in SEQ ID NO 1. Theinferred complete AHAS coding sequence for line CMC31 is listed below asSEQ ID NO 20. The corresponding complete inferred amino acid sequencefor this herbicide resistant AHAS is listed below as SEQ ID NO 21.

EXAMPLES 30-34 PWC16, PWC23, CMC29, WDC33, and WDC38 (ATCC PTA-904,PTA-905, PTA-902, PTA-906, and PTA-908)

Partial cDNA sequences were also determined for the AHAS coding sequencefrom the lines PWC16, PWC23, CMC29, WDC33, and WDC38 (ATCC PTA-904,PTA-905, PTA-902, PTA-906, PTA-908.)

Leaf material from young greenhouse-grown seedlings was ground, andtotal RNA was extracted using the “Plant RNEASY Total RNA Midi kit” fromQiagen, following the manufacture's suggested protocols. Next mRNA waspurified from the extracted total RNA using Qiagen's “Oligotex mRNApurification system,” following the manufacture's suggested protocols.

PCR primers were designed based on an analysis of known AHAS codingsequences from other species. Primers were chosen to correspond tohighly conserved regions with low codon degeneracy. These primers werethen used in PCR amplification of segments of the sequence from RT-PCRreactions from the isolated rice mRNA.

The mRNA was reverse-transcribed using an oligo-dT primer supplied withLife Technologies' “Superscript First-Strand Synthesis System forRT-PCR.” The manufacturer's suggested protocols were followed.

Two primers amplifying approximately 300-350 base pairs were used toamplify the 3′ end of the AHAS coding sequence. The resulting DNAfragments were analyzed by agarose gel electrophoresis and were clonedinto Topo-TA™ vectors, and a number of individual isolates weresequenced. Sequencing was conducted according to standard protocols,using a Perkin Elmer ABI Prism 310 or a Beckman CEQ2000 automatedsequencer. The resulting DNA sequence information was analyzed bycommercially available DNA software analysis programs such asSequencer™.

The observed sequences for lines PWC16, PWC23, CMC29, WDC33, and WDC38are given below as SEQ ID NOS 9 through 13, respectively. Note that inthe wild-type (Cypress) AHAS sequence (SEQ ID NO 14), the codoncorresponding to amino acid 627 is AGT, which encodes serine. In each ofthe sequences of lines PWC16, PWC23, CMC29, WDC33, WDC38 (SEQ ID NOS9-13), the codon corresponding to amino acid 627 is AAT, which encodesasparagine. This serine-asparagine substitution is believed to beresponsible for the herbicide resistance displayed by the AHAS enzyme ofthese lines.

Since the source of the herbicide resistance mutation has beenidentified in the lines PWC16, PWC23, CMC29, WDC33, and WDC38, it isreasonable to infer that the complete coding sequences for each of theselines is the same as that of the “parent” wild type Cypress AHAS (SEQ IDNO 14), except for replacing the AGT codon for serine at amino acidposition 627 with an AAT codon for asparagine. The inferred completeAHAS coding sequence for each of lines PWC16, PWC23, CMC29, WDC33, andWDC38 is listed below as SEQ ID NO 18. The corresponding completeinferred amino acid sequence for this herbicide resistant AHAS is listedbelow as SEQ ID NO 19.

Because the different lines PWC16, PWC23, CMC29, WDC33, and WDC38 havedemonstrated different levels of herbicide tolerance in the field, itwas surprising that the mutations identified for these five lines wereall identical. The sequencing of the AHAS coding sequences of theselines will be repeated for verification. In the event that thisre-checking demonstrates that one or more of these lines in fact has amutation in the AHAS coding sequence other than (or in addition to) thatleading to the Ser-Asn substitution at amino acid 627, any suchmutations are also considered to be within the scope of the presentinvention.

EXAMPLE 35 Wild Type Parent (Cypress); and Comparison of Wild Type AHASSequences from Different Rice Varieties

The AHAS coding sequence for the wild-type “parent” Cypress is givenbelow as SEQ ID NO 14. The inferred amino acid sequence for thewild-type (Cypress) AHAS sequence is listed below as SEQ ID NO 17,corresponding to the translation of the open reading frame of SEQ ID NO14.

Using BLAST to compare sequences, it was found that there is about 89%identity at the amino acid level between wild-type rice AHAS (SEQ ID NO17) and the AHAS sequence for Zea mays reported as GenBank accessionnumber X63553 (576 amino acids of 641).

It is also interesting to compare the wild type AHAS coding sequence forthe rice cultivar Cypress to the wild type AHAS coding sequence reportedby T. Shimizu et al., “Oryza sativa ALS mRNA for acetolactate synthase,complete cds, herbicide sensitive wild type,” BLAST accession numberAB049822. The two sequences are 98% identical at the nucleotide level(1955 bases of 1986). The inferred amino acid sequences are 99%identical (640 amino acids of 644). The vast majority of the 31 basesthat differ between the two nucleotide sequences are “silent” mutations,i.e., they result in different codons that encode the same amino acids.The “non-silent” differences occur at amino acid 11 (Cypress, Thr;Kinmaze, Ala); amino acid 293 (Cypress, Arg; Kinmaze, Trp); amino acid401 (Cypress, Asp; Kinmaze, Gln); and amino acid 643 (Cypress, Met;Kinmaze, Val).

EXAMPLE 36 CMC31 (ATCC PTA-903)

The AHAS coding sequence from line CMC31 was sequenced again, by adifferent set of workers from those who derived SEQ ID NO 1, using theprotocol described below.

1.0 g of fresh tissue from young greenhouse-grown seedlings was ground,and genomic DNA was isolated using the DNeasy Plant Maxi Kit (fromQiagen), following the manufacturer's recommended protocols. The amountof isolated DNA was measured by absorbance reading at 260 nm. 100 ng ofDNA was used in a PCR reaction designed to amplify the 3′ end of theAHAS coding sequence. The following primers, based on the publishedsequence of Oryza sativa var. Kinmaze (GenBank AB049822), were used toamplify the 3′ end of the coding sequence: primer RA7,AGTGGCTGTCTTCGGCTGGTCT (SEQ ID NO 22), and primer RA5,CCTACCACTACCGTCCTGACACAT (SEQ ID NO 23).

The Expand High Fidelity PCR kit from Roche was used to amplify the DNA.DNA from the PCR reaction was checked for correct size using 1% agarosegel electrophoresis. Bands of the expected size were cut from the gel,and the DNA was then purified using the QIAquick Gel Extraction Kit(Qiagen) according to the manufacturer's instructions. Purified DNA wasligated to pCR2.1-TOPO™ vector DNA. After ligation, the DNA wastransformed into One Shot™ competent E. coli cells. Cells were plated,and grown overnight. The following day single colonies were picked,re-plated and numbered.

E. coli cells were lysed by adding a small quantity of cells to 20 ml ofwater, and heating to 95° C. for 5 minutes. PCR was then performed onthe lysed cells with the primers RA7 and RA5 and Taq polymerase.Colonies that were positive for the inserted DNA were grown overnight,and DNA from these cultures was prepared using the Miniprep Plasmid kitfrom Qiagen. DNA was checked again by digestion with the restrictionenzymes PstI and HindIII.

DNA was sequenced by the Big Dye terminator method (Applied Biosystems,Foster City, Calif.). Primer RA7 was used in the sequencing reaction (inthe forward direction). Sequencing was performed on a 377 Perkin Elmerinstrument. Sequences were analyzed using Vector NTI Suite 6.

The resulting sequence is given below as SEQ ID NO 16. This time, thesequence showed the same, single nucleotide G-A substitution, resultingin the same Ser-Asn substitution at amino acid position 627, as was seenfor each of the lines PWC16, PWC23, CMC29, WDC33, WDC38.

The reason for the discrepancy between SEQ ID NO 1 and SEQ ID NO 16 forline CMC31 is currently unknown. As of the filing date of thisapplication, the scenario that is considered most likely is that thedeletion/frame shift mutation shown in SEQ ID NO 1 is correct, and thatthe Ser-Asn substitution implied by SEQ ID NO 16 resulted from somecurrently unknown experimental error, for example mis-labeling of apacket of seed. It is also possible, although it is currently consideredless likely, that line CMC31 does have the same Ser-Asn substitutionseen in the other lines. Repeated sequencing of the AHAS coding sequencefrom seed that is confirmed to be from line CMC31 will clarify thisdiscrepancy.

EXAMPLE 37 WDC37 (ATCCPTA-907)

DNA from line WDC37 was isolated, cloned and sequenced as describedabove for CMC31 (namely, the method using primers RA5 and RA7), exceptthat the DNA from line WDC37 was sequenced by automated dideoxysequencing, generally according to the method of Sanger et al., Proc.Natl. Acad. Sci. USA, vol. 74, pp. 5463-5467 (1977). Reactions wereperformed with the Applied Biosystems (Foster City, Calif.) Prism BigDye terminator cycle sequencing kit with AmpliTaq DNA polymerase FS andwere electrophoresed on an Applied Biosystems Prism 377 DNA sequencer.

To date, the mutation responsible for herbicide resistance in WDC37 hasnot been identified. The sequences obtained to date have beenindistinguishable from wild type. Possible reasons for this includemis-marked seed, segregation effects causing wild type AHAS sequences tooccasionally be picked up in the herbicide resistant line, or a mutationin a different part of the AHAS molecule from that targeted by the PCRprimers RA5 and RA7. Repetitions of these sequencing efforts, usingfresh seed confirmed to be from WDC37, and including the entire AHAScoding sequence will clarify this discrepancy.

FURTHER EXAMPLES

The AHAS coding sequence for ATCC No. 97523 (93AS3510) is given below asSEQ ID NO 24. The inferred amino acid sequence for the ATCC No. 97523AHAS is listed below as SEQ ID NO 25, corresponding to the translationof the open reading frame of SEQ ID NO 24. Note that in the wild-type(Cypress) AHAS sequence (SEQ ID NO 14), the codon corresponding to aminoacid 628 is GGG, which encodes glycine. In the sequence of line 97523(SEQ ID NO 24), the codon corresponding to amino acid 628 (in SEQ ID NO25) is GAG, which encodes glutamic acid. This glycine-glutamic acidsubstitution is believed to be responsible for the herbicide resistancedisplayed by the AHAS enzyme of the ATCC No. 97523 line.

The complete DNA sequences and inferred amino acid sequences of the AHASmolecules for each of the rice plants having ATCC Accession Nos. 203419,203420, 203421, 203422, 203423, 203424, 203425, 203426, 203427, 203428,203429, 203430, 203431, 203432, 203433, PTA-904, PTA-905, PTA-902,PTA-906, PTA-907, and PTA-908 will also be determined (or redetermined)using generally similar protocols, or other techniques well known in theart.

Cloning into Other Green Plants.

These and other cloned, herbicide-resistant AHAS nucleotide sequencesfrom rice plants may be used to transform herbicide resistance intoother rice plants, as well as transforming other green plants generallyto impart herbicide resistance. Herbicide resistance may be introducedinto other rice plants, for example, either by traditional breeding,back-crossing, and selection; or by transforming cultivars with thecloned resistant AHAS nucleotide sequences. Direct transformation ofrice cultivars has the potential to allow quick introduction of theherbicide resistance characteristics into a variety, without requiringmultiple generations of breeding and back-crossing to attain uniformity.

Furthermore, at least in the case of rice transformed via the preferredvector of U.S. Pat. No. 5,719,055, much of the concern that someindividuals have expressed over the propriety of the genetictransformation of agricultural species should be alleviated. Thetransformation of a rice variety with a nucleotide sequence from anotherrice plant would only speed up what could also be accomplished throughtraditional breeding techniques. No coding sequences exogenous to riceplants would be introduced, merely the efficient introduction of a riceAHAS allele from another rice plant. The use of the vector of U.S. Pat.No. 5,719,055 allows the introduction of the desired coding sequenceonly, without any other coding sequences being introduced into thegenome. No antibiotic-resistance genes or other markers will be needed:selection for successful transformation events can be based directly onthe herbicide resistance itself. As explained more fully in U.S. Pat.No. 5,719,055, the only sequences that need be introduced in addition tothe nucleotide sequence of interest are flanking insertion sequencesrecognized by the transposase used by the vector. The insertionsequences are not themselves coding sequences, and are inert in theabsence of the transposase; furthermore, the vector is designed so thatthe transposase is not encoded by any DNA that is inserted into thetransformed chromosome. The only portion of the transformed DNA thatwill be active following transformation is the resistant AHAS nucleotidesequence itself. That AHAS nucleotide sequence is derived from rice, sothe transformed rice plants will not be “transgenic” in the usual senseof carrying coding DNA from another species.

It will be understood by those skilled in the art that the nucleic acidsequences of the resistant mutant AHAS enzymes from ATCC Accession Nos.97523, 203419, 203420, 203421, 203422, 203423, 203424, 203425, 203426,203427, 203428, 203429, 203430, 203431, 203432, 203433, PTA-904,PTA-905, PTA-902, PTA-903, PTA-906, PTA-907, and PTA-908 are not theonly sequences that can be used to confer resistance. Also contemplatedare those nucleic acid sequences that encode identical proteins butthat, because of the degeneracy of the genetic code, possess differentnucleotide sequences. The genetic code may be found in numerousreferences concerning genetics or biology, including, for example, FIG.9.1 on page 214 of B. Lewin, Genes VI (Oxford University Press, NewYork, 1997). FIG. 9.3 on page 216 of Lewin directly illustrates thedegeneracy of the genetic code. For example, the codon for asparaginemay be AAT or AAC.

The sequences may be transformed into a plant of interest with orwithout some or all of the native Oryza sativa AHAS gene's introns,which may be identified by means well known in the art. Introns canaffect the regulation of gene expression.

The expression products are preferably targeted to the chloroplasts,which are believed to be the major site for wild type AHAS activity ingreen plants. The targeting signal sequence normally corresponds to theamino terminal end of the protein expression product, and thecorresponding coding sequence should therefore appear upstream of the 5′end of the coding sequence. This targeting is preferably accomplishedwith the native Oryza sativa AHAS signal sequence, but it may also useother plant chloroplast signal sequences known in the art, such as, forexample, those disclosed in Cheng et al., J. Biol. Chem., vol. 268, pp.2363-2367 (1993); see also Comai et al., J. Biol. Chem., vol. 263, pp.15104-15109 (1988).

The invention also encompasses nucleotide sequences encoding AHASproteins having one or more silent amino acid changes in portions of themolecule not involved with resistance or catalytic function. Forexample, alterations in the nucleotide sequence that result in theproduction of a chemically equivalent amino acid at a given site arecontemplated; thus, a codon for the amino acid alanine, a hydrophobicamino acid, may be substituted by a codon encoding another hydrophobicresidue, such as glycine, or may be substituted with a more hydrophobicresidue such as valine, leucine, or isoleucine. Similarly, changes thatresult in the substitution of one negatively-charged residue foranother, such as aspartic acid for glutamic acid, or onepositively-charged residue for another, such as lysine for arginine, canalso be expected to produce a biologically equivalent product. See,e.g., FIG. 1.8 on page 10 of Lewin (1997), showing the nature of theside chains of the “standard” 20 amino acids encoded by the geneticcode. (Note also a typographical error in that published figure, namelythat the abbreviation for glutamine should be “Gln.”)

The invention also encompasses chimeric nucleotide sequences, in whichthe mutated portion of a resistant rice AHAS nucleotide sequence isrecombined with unaltered portions of the AHAS nucleotide sequence fromanother species.

This invention relates not only to a functional AHAS enzyme having theamino acid sequence encoded by a mutant, resistant AHAS nucleotidesequence described in this specification, including for example thosefrom one of the identified rice plants deposited with ATCC, but also toan enzyme having modifications to such a sequence resulting in an aminoacid sequence having the same function (i.e., a functional AHAS enzyme,with resistance to at least some herbicides that normally interfere withAHAS), and about 60-70%, preferably 90% or greater homology to thesequence of the amino acid sequence encoded by the AHAS nucleotidesequence of at least one such ATCC-deposited rice line, more preferablyabout 95% or greater homology, particularly in conserved regions suchas, for example, a putative herbicide binding site. “Homology” meansidentical amino acids or conservative substitutions (e.g., acidic foracidic, basic for basic, polar for polar, nonpolar for nonpolar,aromatic for aromatic). The degree of homology can be determined bysimple alignment based on programs known in the art, such as, forexample, GAP and PILEUP by GCG, or the BLAST software available throughthe NIH internet site. Most preferably, a certain percentage of“homology” would be that percentage of identical amino acids.

A particular desired point mutation may be introduced into an AHAScoding sequence using site-directed mutagenesis methods known in theart. See, e.g., R. Higuchi, “Recombinant PCR,” pp. 177-183 in M. Inniset al. (Eds.), PCR Protocols: A Guide to Methods and Applications,Academic Press (1990); U.S. Pat. No. 6,010,907; Kunkel, Proc. Natl.Acad. Sci. USA, vol. 82, pp. 488-492 (1985); Kunkel et al., MethodsEnzymol., vol. 154, pp. 367-382 (1987); U.S. Pat. No. 4,873,192; Walkeret al. (Eds.), Techniques in Molecular Biology (MacMillan, New York,1983); or the Genoplasty™ protocols of ValiGen (Newtown, Pa.).

Isolated AHAS DNA sequences of the present invention are useful totransform target crop plants, and thereby confer resistance. A broadrange of techniques currently exists for achieving the direct orindirect transformation of higher plants with exogenous DNA, and anymethod by which one of the novel sequences can be incorporated into thehost genome, and stably inherited by its progeny, is contemplated by thepresent invention.

The cloned AHAS coding sequence should be placed under the control of asuitable promoter, so that it is appropriately expressed in cells of thetransformed plant. It is expected that the most suitable promoter wouldbe a native AHAS promoter. The native AHAS promoter could be that of anyplant, for example, the native AHAS promoter from the plant that isbeing transformed. Alternatively, it is expected that the native riceAHAS promoter will function appropriately in other green plantsgenerally (including, e.g., both monocots and dicots), and that forsimplicity the same rice AHAS promoter may be used when transforming anyplant species of interest.

The native AHAS promoter, whether that from rice or from another plant,may be isolated as follows. AHAS cDNA is isolated and amplified, forexample by PCR or by cloning into a bacterium such as E. coli orAgrobacterium, and is denatured into single-stranded DNA. A genomiclibrary is prepared for rice or other plant of interest using standardtechniques, and Southern blotting is used to hybridize segments ofgenomic DNA to the AHAS cDNA. The hybridizing DNA segments are amplifiedthrough PCR and sequenced. The promoter will be found in segmentsupstream of the transcription initiation site.

Note that it is not necessary to identity the sequence of the AHASpromoter precisely. Where the upstream sequence that constitutes thepromoter has not been precisely identified, the promoter willnevertheless be included by taking a sufficiently large number of bases“upstream” of the transcription initiation site. The fact that “extra”bases may also be included in addition to the promoter is acceptable.The number of upstream bases needed to encompass a particular promotermay readily be determined in a particular case, and for the reasons justgiven, the precise number of bases is not crucial. In general, sequencesof about 500, 1000, or 1500 bases upstream from the transcriptioninitiation site should suffice in most cases.

As a further alternative, a constitutive promoter could be used tocontrol the expression of the transformed mutant AHAS coding sequence.Promoters that act constitutively in plants are well known in the art,and include, for example, the cauliflower mosaic virus 35S promoter.

Transformation of plant cells can be mediated by the use of vectors. Acommon method for transforming plants is the use of Agrobacteriumtumefaciens to introduce a foreign nucleotide sequence into the targetplant cell. For example, a mutant AHAS nucleotide sequence is insertedinto a plasmid vector containing the flanking sequences in theTi-plasmid T-DNA. The plasmid is then transformed into E. coli. Atriparental mating is carried out among this strain, an Agrobacteriumstrain containing a disarmed Ti-plasmid containing the virulencefunctions needed to effect transfer of the AHAS-containing T-DNAsequences into the target plant chromosome, and a second E. coli straincontaining a plasmid having sequences necessary to mobilize transfer ofthe AHAS construct from E. coli to Agrobacterium. A recombinantAgrobacterium strain, containing the necessary sequences for planttransformation, is used to infect leaf discs. Discs are grown onselection media and successfully transformed regenerants are identified.The recovered plants are resistant to the effects of herbicide whengrown in its presence.

Plant viruses also provide a possible means for transfer of exogenousDNA.

Direct uptake of DNA by plant cells can also be used. Typically,protoplasts of the target plant are placed in culture in the presence ofthe DNA to be transferred, along with an agent that promotes the uptakeof DNA by protoplasts. Such agents include, for example, polyethyleneglycol and calcium phosphate.

Alternatively, DNA uptake can be stimulated by electroporation. In thismethod, an electrical pulse is used to open temporary pores in aprotoplast cell membrane, and DNA in the surrounding solution is thendrawn into the cell through the pores. Similarly, microinjection can beused to deliver the DNA directly into a cell, preferably directly intothe nucleus of the cell.

In many of these techniques, transformation occurs in a plant cell inculture. Subsequent to the transformation event, plant cells must beregenerated to whole plants. Techniques for the regeneration of matureplants from callus or protoplast culture are known for a large number ofplant species. See, e.g., Handbook of Plant Cell Culture, Vols. 1-5,1983-1989 McMillan, N.Y.

Alternate methods are also available that do not necessarily require theuse of isolated cells and plant regeneration techniques to achievetransformation. These are generally referred to as “ballistic” or“particle acceleration” methods, in which DNA-coated metal particles arepropelled into plant cells by either a gunpowder charge (see Klein etal., Nature 327: 70-73, 1987) or by electrical discharge (see EPO 270356). In this manner, plant cells in culture or plant reproductiveorgans or cells, e.g. pollen, can be stably transformed with the DNAsequence of interest.

In certain dicots and monocots, direct uptake of DNA is the preferredmethod of transformation. For example, in maize or rice the cell wall ofcultured cells is digested in a buffer with one or more cellwall-degrading enzymes, such as cellulase, hemicellulase, and pectinase,to isolate viable protoplasts. The protoplasts are washed several timesto remove the degrading enzymes, and are then mixed with a plasmidvector containing the nucleotide sequence of interest. The cells can betransformed with either PEG (e.g. 20% PEG 4000) or by electroporation.The protoplasts are placed on a nitrocellulose filter and cultured on amedium with embedded maize cells functioning as feeder cultures. After2-4 weeks, the cultures in the nitrocellulose filter are placed on amedium containing herbicide and maintained in the medium for 1-2 months.The nitrocellulose filters with the plant cells are transferred to freshmedium with herbicide and nurse cells every two weeks. Theun-transformed cells cease growing and die after a time.

Other methods of transforming plants are described in B. Jenes et al,and in S. Ritchie et al., in S.-D. Kung et al. (Eds.), TransgenicPlants, vol. 1, Engineering and Utilization, Academic Press, Inc.,Harcourt Brace Jovanovich (1993); and in L. Mannonen et al., CriticalReviews in Biotechnology, vol. 14, pp. 287-310 (1994). See also thevarious references cited on pages 15-17 of published internationalpatent application WO 00/26390, each of which is incorporated byreference.

A particularly preferred transformation vector, which may be used totransform seeds, germ cells, whole plants, or somatic cells of monocotsor dicots, is the transposon-based vector disclosed in U.S. Pat. No.5,719,055. This vector may be delivered to plant cells through one ofthe techniques described above or, for example, via liposomes that fusewith the membranes of plant cell protoplasts.

The present invention can be applied to transform virtually any type ofgreen plant, both monocot and dicot. Among the crop plants and otherplants for which transformation for herbicide resistance is contemplatedare (for example) rice, maize, wheat, millet, rye, oat, barley, sorghum,sunflower, sweet potato, casava, alfalfa, sugar cane, sugar beet, canolaand other Brassica species, sunflower, tomato, pepper, soybean, tobacco,melon, lettuce, celery, eggplant, carrot, squash, melon, cucumber andother cucurbits, beans, cabbage and other cruciferous vegetables,potato, tomato, peanut, pea, other vegetables, cotton, clover, cacao,grape, citrus, strawberries and other berries, fruit trees, and nuttrees. The novel sequences may also be used to transform turfgrass,ornamental species, such as petunia and rose, and woody species, such aspine and poplar.

Enzyme Purification, Analysis, and Sequencing

Preliminary data (reported below) suggest that rice may produce at leastthree different AHAS isozymes, which would presumably be encoded bydifferent AHAS nucleotide sequences in the rice genome, although theexperiments completed as of the filing date of this application do notyet rule out the possibility that different isozymes could be encoded bya single gene, but produced by separate pathways such as alternativesplicing of mRNA, or alternative post-translational processing ofpolypeptides. Incidentally, it is believed that the present patentapplication is the first published report that rice appears to have atleast three different AHAS isozymes. To the inventor's knowledge, it hadnot previously been reported that rice had more than one form of theAHAS enzyme.

The sequence obtained for the first isozyme will be used to prepareprimers for amplifying and sequencing the other isozymes, either fromgenomic DNA or from cDNA. Greater levels of resistance may be obtainedin plants carrying resistant alleles in multiple AHAS nucleotidesequences. Such plants may readily be bred by crossing and backcrossingthrough means known in the art, or by site-directed mutagenesis. Evengreater levels of resistance may be obtained by crossing such a “doublemutant” or “triple mutant” with the metabolic-based herbicide resistantrice lines disclosed in U.S. Pat. No. 5,545,822, as typified by the ricehaving ATCC accession number 75295, as discussed earlier, since itsmetabolic resistance is based on a separate, currently unknownmechanism.

The preliminary isozyme data reported below suggests that the resistanceseen in some of the novel lines may have resulted from mutations indifferent AHAS isozymes. For example, in the non-resistant parentalCypress line, the putative AHAS-1 isozyme (defined below) appeared toaccount for about 25% of the total activity, while putative AHAS-2 andputative AHAS-3 together appeared to account for about 75% of totalactivity. By contrast, in ATCC 97523, putative AHAS-1 appeared toaccount for about 75% of total activity, with putative AHAS-2 andputative AHAS-3 together appearing to account for about 25% of totalactivity; while in PWC23 putative AHAS-1 appeared to account for about95% of total activity, with putative AHAS-2 and putative AHAS-3 togetherappearing to account for about 5% of total activity.

It is hypothesized that the amino acid end-products of the processcatalyzed by AHAS-1 may cause feedback suppression of AHAS-2 and AHAS-3in the lines with low activities in the latter two isozymes.

In addition, total AHAS activity in many of the novel resistant linesexceeded that in the non-resistant Cypress parent.

Preliminary Note: It is believed that rice possesses multiple AHASisozymes, perhaps two or three such isozymes. The procedures reportedunder the present heading (“Enzyme Purification, Analysis, andSequencing”) were conducted to attempt to separate the differentisozymes. Since these experiments were conducted, it has come to theinventor's attention (B. J. Singh, private communication) that theseparticular isozyme separation procedures may be subject to artifacts, toexperimental errors. As of the filing date of the present internationalPatent Cooperation Treaty application, this question had not beenresolved to the inventor's satisfaction. While it is believed that ricedoes have multiple AHAS isozymes, the particular experimental datareported in this section, concerning enzyme purification and separation,may or may not be a reliable indication of that AHAS isozyme activity.Should these particular experimental data turn out not to be reliable,then other enzymatic purification and separation procedures known in theart may be substituted. Furthermore, it is important to note that theother results reported in this patent application do not depend uponwhether the isozyme data reported in the present section are correct orincorrect.

The procedures used to separate the acetohydroxyacid synthase isozymesin rice from one another were substantially as described in B. Singh etal., “Separation and Characterization of Two Forms of AcetohydroxyacidSynthase from Black Mexican Sweet Corn Cells,” J. Chromatogr., vol. 444,pp. 251-261 (1988). Suspension cells, or shoot tissues fromgreenhouse-grown plants at the 3-4 leaf stage of development, were usedfor crude enzyme extraction. For extraction from suspension cells, 16grams of cells were harvested 8 days after subculturing of Cypresssuspension cultures. Following crude enzyme pelleting, the enzyme wasre-suspended in 25 mM potassium phosphate buffer (pH=7.0) containing 5mM pyruvate, 5 mM EDTA, and 5 μM FAD (flavin adenine dinucleotide). Theisozymes were then separated by HPLC on a Waters 600 chromatograph(Amersham Pharmacia Biotech, Piscataway, N.J.) at an eluent flow rate of1 mL per minute. Following filtration through a 0.45 μm Millex(Millipore, Bedford, Mass.) syringe filter, 2.00 mL of each sample wasloaded onto a Mono Q HR 5/5 column (5×0.5 cm) (Amersham PharmaciaBiotech, Piscataway, N.J.) that had been pre-equilibrated with the samebuffer. After injection and elution of 5 mL of eluent, a linear20-minute gradient of 0-0.5 M potassium chloride in equilibration bufferwas initiated. One-mL fractions were collected and assayed for AHASenzyme activity. Additional purification procedures that are standard inthe art may optionally be used, such as gel electrophoresis oradditional HPLC separations.

Several rice lines have been assayed in this manner. The remainingresistant lines will be assayed in the same manner. Each of the sampledlines appeared to have at least three AHAS isozymes. Differences werenoted among the lines with respect to total AHAS enzyme activity, theoverall level of herbicide resistance, and the activity of theindividual isozymes.

Table 9 depicts qualitatively the relative activities seen in severallines for the three putative isozymes that have been tentativelydesignated “AHAS-1,” “AHAS-2,” and “AHAS-3.” (The numbering correspondsto the order in which the enzymes eluted from the HPLC.)

TABLE 9 Relative AHAS Isozyme Activity, reported qualitatively on a5-point scale, based on absorbance at 520 nm Cypress ATCC 97523 PWC16PWC23 CMC29 CMC31 WDC33 AHAS-1 1 2  0 5 3   0+ 0+ AHAS-2 1 0+ 1 0 0+ 10+ AHAS-3 2 0+  1+  0+ 1− 2 0+

To prepare isolated AHAS isozymes for direct amino acid sequencing, thesame protocols were used as described above, except that aliquots werecombined from four separate runs through the HPLC (4 injections, 2mL/injection). The combined aliquots were concentrated by coolevaporation of the liquid using a Savant Instruments SpeedVac(Farmingdale, N.Y.). The concentrated samples were then run through aPD-10 Sephadex G-25 (Amersham Pharmacia Biotech, Piscataway, N.J.)desalting column prior to freeze-drying. The amino acid sequence of eachof the three isozymes will be determined by Edman degradation. (The4×1.0 mL=4.0 mL combined aliquot having the highest peak for eachisozyme was selected for amino acid sequencing, on the assumption thatthis aliquot had a relatively high and a relatively pure concentrationof the particular isozyme.)

The Edman degradation technique and related protein analysis techniquesare well known in the peptide art. Briefly, the amino-terminal residueof a polypeptide is labeled and cleaved from the peptide withoutdisrupting the peptide bonds between the other amino acid residues. TheEdman degradation sequentially removes one residue at a time from theamino end of a polypeptide. Phenyl isothiocyanate reacts with theuncharged terminal amino group of the peptide to form aphenylthiocarbamoyl derivative. Under acidic conditions, a cyclicderivative of the terminal amino acid is liberated, which leaves anintact peptide shortened by one amino acid. The cyclic compound is aphenylthiohydantoin (PTH)-amino acid. The PTH-amino acid is thenidentified by standard procedures. See generally R. Meyers (ed.),Molecular Biology and Biotechnology, pp. 731-741, and 764-773 (1995).

The polypeptide sequencing results will be used to complement theresults from the mRNA RT-PCR sequencing described previously. The aminoacid sequences will be confirmed by designing DNA primers from theobserved amino acid partial sequences, cloning the AHAS nucleotidesequences from genomic or cDNA libraries of the wild type and mutantrice lines, and sequencing the nucleotide sequences thus cloned. Thesequences will also be confirmed by comparing the complementarity of thesequences determined for the positive strands and the negative strandsof each.

The amino acid sequence data will be used to design primers for PCRamplification of the putative AHAS isozymes, along with the data fromthe known AHAS coding sequence (e.g. SEQ ID NO. 14). Also, it ispossible through standard techniques to digest the protein into smallerpieces, which are then sequenced individually to give amino acidsequences for internal regions of the protein.

Proposed Mechanisms of Action

Preliminary Note: The discussion under the present heading (“ProposedMechanisms of Action”) is subject to the same caveats mentioned underthe previous heading concerning the isozyme purification and separationprotocols that have been used to date. The validity of the other datareported here do not depend on these proposed mechanisms of action.

Without wishing to be bound by the theory presented in this section, thefollowing hypotheses are consistent with the preliminary isozyme datadescribed under the previous heading: Rice appears to have at leastthree AHAS isozymes, isozymes that are normally produced by a rice plantat different activity levels. Except for ATCC 75295, the mutationsdiscovered by the present inventor for herbicide resistance in riceappear to be mutations in the AHAS enzymes themselves, rather thanmutations arising from another source such as a mutation in a metabolicpathway or a mutation in the regulation of an AHAS gene. In some of theresistant mutants, at least in the absence of herbicide, the relativeactivity levels of the three AHAS isozymes appear not to besubstantially altered compared to wild-type, perhaps because theresistance mutation in these lines appears in the “predominant” isozyme.In at least one of the resistant mutants, the relative activities of allthree isozymes appeared to be lower than wild-type.

On the other hand, there are resistant mutants in which one of the“alternate” isozymes appears to be preferentially produced, for unknownreasons. The increased activity of this mutant isozyme causes feedbackinhibition of the other two isozymes; i.e., higher levels of amino acidproducts resulting from the activity of the mutant isozyme inhibit theexpression of the other two isozymes through normal regulatory feedbackmechanisms.

The herbicide appears to act by strongly (or even irreversibly) bindingto the AHAS molecule. This binding does not completely eliminate AHASactivity, even in wild-type AHAS, although herbicide binding does reducethe activity (˜50%) sufficiently to kill wild-type plant cells. Thereappears to be a “saturation” point in the herbicide concentration, e.g.˜10 μM (depending on the herbicide), above which activity of wild-typeenzyme does not decrease substantially.

By contrast, even at much higher herbicide concentrations, e.g., even upto ˜1000 μM, the activity of some of the mutant AHAS enzymes is stillroughly comparable to the activity of wild-type enzyme in the absence ofany herbicide, suggesting that the mutant AHAS enzymes do not bind theherbicides as strongly. Yet even those mutant AHAS enzymes that normallyshow strong resistance to AHAS-acting herbicides are neverthelesssusceptible to some of the AHAS-acting herbicides. This propertysuggests that the resistance of that enzyme to certain herbicides is dueto a weaker binding affinity between those herbicides and the mutantenzyme; and not, for example, to pre-herbicide overproduction of theAHAS-catalyzed amino acids that allows the plant cells to survive on“reserves” of those amino acids until the effect of the herbicide hasworn off. Were such an overproduction responsible for the herbicideresistance, then across-the-board resistance to all AHAS-actingherbicides would be expected.

Miscellaneous

Through routine breeding practices known in the art, progeny will bebred from each of the resistant parent rice lines identified above. Onceprogeny are identified that are demonstrably resistant, those progenywill be used to breed varieties for commercial use. Crossing andback-crossing resistant plants with other germplasm through standardmeans will yield herbicide-resistant varieties and hybrids having goodproductivity and other agronomically desirable properties.Alternatively, direct transformation into a variety or into a parent ofa hybrid having agronomically desirable properties may be employed, asdirect transformation can accelerate the overall selection and breedingprocess.

Because red rice and commercial rice belong to the same species, theplanting of a herbicide-resistant commercial rice crop entails some riskthat herbicide resistance would be transferred to red rice. However,rice is self-pollinating, and the frequency of outcrossing is low, evenbetween immediately adjacent plants flowering in synchrony. Thelikelihood of transferring resistance to red rice could be minimized bybreeding resistant varieties that flower significantly earlier than doesred rice (e.g., using conventional breeding techniques, or by tissueculture such as another culture). Maintaining an early-maturingphenotype in resistant varieties, for example, will be desirable toreduce the likelihood of outcrossing to red rice. In addition, breedinghigher levels of resistance (e.g., by crossing lines with different AHASisozymes with one another, or crossing lines with resistant AHAS enzymeswith the metabolic resistance of ATCC 75295) will allow control of theoutcrossed red rice by applying higher herbicide rates than theoutcrossed red rice will tolerate.

If a strain of red rice should nevertheless develop that is resistant tothe same herbicides as resistant commercial rice, the plants can alwaysbe treated with a broad range of other available herbicides—particularlyif the resistant red rice were discovered early, before having muchopportunity to propagate.

The same or analogous techniques should be employed to inhibit theout-crossing of herbicide resistance into “weedy” or wild relatives ofother crop species.

Because each of the herbicides tested inhibits the activity ofacetohydroxyacid synthase, and because resistance to each of theseherbicides has been demonstrated in the novel lines, it is expected thatthe novel herbicide resistant rice will show resistance to otherherbicides that normally inhibit this enzyme. In addition to thosediscussed above, such herbicides include others of the imidazolinone andsulfonylurea classes, including at least the following: primisulfuron,chlorsulfuron, imazamethabenz methyl, and triasulfuron. Other classes ofAHAS herbicides known in the art include triazolopyrimidines,triazolopyrimidine sulfonamides, sulfamoylureas, sulfonylcarboxamides,sulfonamides, pyrimidyloxybenzoates, phthalides, pyrimidylsalicylates,carbamoylpyrazolines, sulfonylimino-triazinyl heteroazoles, N-protectedvalylanilides, sulfonylamide azines, pyrimidyl maleic acids,benzenesulfonyl carboxamides, substituted sulfonyldiamides, andubiquinone-o.

As used in the specification and claims, the term “mutation-inducingconditions” refers to conditions that will cause mutations in a plant'sgenome at rates substantially higher than the background rate. A varietyof such conditions are well-known to those in the art. They include, forexample, exposing seeds to chemical mutagens or ionizing radiation aspreviously described. Such conditions also include growing cells intissue culture (another culture, callus culture, suspension culture,protoplast culture, etc.), with or without deliberately exposing thecells to additional mutation-inducing conditions other than those thatare inherent in tissue culture. (It is known that tissue culture is perseconducive to the production of genetic variability, includingmutations.) Depending on the particular mutation-inducing conditionsused, mutations may best be induced at different stages in the lifecycle, e.g., with dry seeds, with pre-germinated seeds, etc.

As used in the specification and claims, the term “imidazolinone” meansa herbicidal composition comprising one or more chemical compounds ofthe imidazolinone class, including by way of example and not limitation,2-(2-imidazolin-2-yl)pyridines, 2-(2-imidazolin-2-yl)quinolines and2-(2-imidazolin-2-yl) benzoates or derivatives thereof, including theiroptical isomers, diastereomers and/or tautomers exhibiting herbicidalactivity, including by way of example and not limitation2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-3-quinolinecarboxylicacid (generic name imazaquin);2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-5-ethyl-3-pyridinecarboxylicacid (generic name imazethapyr); and2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-5-(methoxymethyl)-3-pyridinecarboxylicacid (generic name imazamox);2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-3-pyridinecarboxylicacid (generic name imazapyr);2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-5-methyl-3-pyridinecarboxylicacid) (generic name imazameth, also known as imazapic); and the otherexamples of imidazolinone herbicides given in the specification.

As used in the specification and claims, the term “sulfonylurea” means aherbicidal composition comprising one or more chemical compounds of thesulfonylurea class, which generally comprise a sulfonylurea bridge,—SO₂NHCONH—, linking two aromatic or heteroaromatic rings, including byway of example and not limitation 2-(((((4,6-dimethoxypyrimidin-2-yl)aminocarbonyl))aminosulfonyl))-N,N-dimethyl-3-pyridinecarboxamide(generic name nicosulfuron);3-[4,6-bis(difluoromethoxy)-pyrimidin-2-yl]-1-(2-methoxycarbonylphenylsulfonyl)urea (generic name primisulfuron);2-[[[[(4,6-dimethyl-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]benzoicacid methyl ester (generic name sulfometuron methyl); methyl2-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl]benzoate(generic name metsulfuron methyl);methyl-2-[[[[N-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)methylamino]carbonyl]amino]sulfonyl]benzoate (generic name tribenuronmethyl); methyl-3-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl]-2-thiophenecarboxylate (generic namethifensulfuron methyl);2-chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminocarbonyl]benzenesulfonamide(generic name chlorsulfuron); ethyl2-[[[[(4-chloro-6-methoxypyrimidin-2-yl)amino]carbonyl]amino]sulfonyl]benzoate(generic name chlorimuron ethyl); methyl2-[[[[N-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)methylamino]carbonyl]amino]sulfonyl]benzoate(generic name tribenuron methyl);3-(6-methoxy-4-methyl-1,3,5-triazin-2-yl)-1-[2-(2-chloroethoxy)-phenylsulfonyl]-urea(generic name triasulfuron); and the other examples of sulfonylureaherbicides given in the specification.

As used in the specification and claims, unless otherwise clearlyindicated by context, the term “plant” is intended to encompass plantsat any stage of maturity, as well as any cells, tissues, or organs takenor derived from any such plant, including without limitation anyembryos, seeds, leaves, stems, flowers, fruits, roots, tubers, singlecells, gametes, another cultures, callus cultures, suspension cultures,other tissue cultures, or protoplasts. Also, unless otherwise clearlyindicated by context, the term “plant” is intended to refer to aphotosynthetic organism or green plant including algae, mosses, ferns,gymnosperms, and angiosperms. The term excludes, however, bothprokaryotes, and eukaryotes that do not carry out photosynthesis such asyeast, other fungi, and the so-called red plants and brown plants thatdo not carry out photosynthesis.

Unless otherwise clearly indicated by context, the “genome” of a plantrefers to the entire DNA sequence content of the plant, includingnuclear chromosomes, mitochondrial chromosomes, chloroplast chromosomes,plasmids, and other extra-nuclear or extra-chromosomal DNA. If, forexample, a herbicide resistance nucleotide sequence is incorporated intothe cells of a transformed plant in a plasmid or other genetic elementthat might not otherwise be consistently maintained and inherited by theplant and its progeny, then the herbicide resistance trait itself may beused to apply selective pressure upon such plants to maintain theherbicide resistance phenotype and genotype. Such a plant is consideredto have the herbicide resistance nucleotide sequence in its “genome”within the contemplation of this definition.

Unless otherwise clearly indicated by context, the “progeny” of a plantincludes a plant of any subsequent generation whose ancestry can betraced to that plant.

Unless otherwise clearly indicated by context, a “derivative” of aherbicide-resistant plant includes both the progeny of thatherbicide-resistant plant, as the term “progeny” is defined above; andalso any mutant, recombinant, or genetically-engineered derivative ofthat plant, whether of the same species or of a different species;where, in either case, the herbicide-resistance characteristics of theoriginal herbicide-resistant plant have been transferred to thederivative plant. Thus a “derivative” of a rice plant with a resistantAHAS enzyme would include, by way of example and not limitation, any ofthe following plants that express the same resistant AHAS enzyme: F₁progeny rice plants, F₂ progeny rice plants, F₃₀ progeny rice plants, atransgenic corn plant transformed with a herbicide resistance nucleotidesequence from the resistant rice plant, and a transgenic sweet potatoplant transformed with a herbicide resistance nucleotide sequence fromthe resistant rice plant.

The following definitions should be understood to apply throughout thespecification and claims, unless otherwise clearly indicated by context.

An “isolated” nucleic acid sequence is an oligonucleotide sequence thatis located outside a living cell. A cell comprising an “isolated”nucleic acid sequence is a cell that has been transformed with a nucleicacid sequence that at one time was located outside a living cell; or acell that is the progeny of, or a derivative of, such a cell.

A “functional” or “normal” AHAS enzyme is one that is capable ofcatalyzing the first step in the pathway for synthesis of the essentialamino acids isoleucine, leucine, and valine; regardless of whether theenzyme expresses herbicide resistance.

A “resistant” plant is one that produces a functional AHAS enzyme, andthat is capable of reaching maturity when grown in the presence ofnormally inhibitory levels of a herbicide that normally inhibits AHAS.The term “resistant” or “resistance,” as used herein, is also intendedto encompass “tolerant” plants, i.e., those plants that phenotypicallyevidence adverse, but not lethal, reactions to one or more AHASherbicides. A “resistant” AHAS enzyme is a functional AHAS enzyme thatretains substantially greater activity than does a wild-type AHAS enzymein the presence of normally inhibitory levels of an AHAS herbicide, asmeasured by in vitro assays of the respective enzymes' activities. A“wild-type” or “sensitive” plant is one that produces a functional AHASenzyme, where the plant is sensitive to normally inhibitory levels of aherbicide that normally inhibits AHAS. A “resistant” plant is a plantthat is resistant to normally inhibitory levels of a herbicide thatnormally inhibits AHAS (either due to a resistant AHAS enzyme or anothermechanism of resistance in the plant). Note that within thecontemplation of this last definition, “wild-type” plants includecultivated varieties; the designation “wild-type” refers to the presenceor absence of normal levels of herbicide sensitivity, and in the contextof this specification and the claims the term “wild-type” carries noconnotation as to whether a particular plant is the product ofcultivation and artificial selection, or is found in nature in anuncultivated state.

A “wild-type” AHAS enzyme or “wild-type” AHAS sequence is an AHAS enzymeor a DNA sequence encoding an AHAS enzyme, respectively, that does notimpart herbicide resistance. Thus, within the scope of this definition,a “wild-type” AHAS may, for example, contain one or more mutations,provided that the mutations do not impart herbicide resistance. In somespecies, such as rice, as was seen for example in the varieties Kinmazeand Cypress, more than one wild-type AHAS may naturally exist indifferent varieties. A “wild-type” AHAS includes, for example, any ofthese multiple AHAS enzymes from different varieties. A “wild-type” AHASalso includes, for example, a hybrid of two or more of these wild-typeAHAS enzymes. (A “hybrid” of different AHAS enzymes corresponds exactlyto at least one of the “parent” enzymes at every amino acid in itssequence; for example, an AHAS that is identical to the Kinmaze AHAS atamino acids 1 through 300, and that is identical to the Cypress AHAS atamino acids 301 through the carboxy terminus.)

The complete disclosures of all references cited in this specificationare hereby incorporated by reference, as are the complete disclosures ofthe present inventor's U.S. provisional patent application Ser. No.60/107,255, filed 5 Nov. 1998; U.S. provisional patent application Ser.No. 60/163,765, filed 5 Nov. 1999; international patent applicationnumber PCT/US99/26062, filed 5 Nov. 1999; and U.S. provisionalapplication Ser. No. 60/203,434, filed 10 May 2000. In the event of anotherwise irreconcilable conflict, however, the present specificationshall control. In particular, the preliminary nucleotide sequence datacontained in these prior applications is not incorporated to the extentthat it is superseded by the sequence data contained in the presentspecification.

Notes on herbicide nomenclature—the following listing gives trade names,generic names, and chemical names for various herbicides: Pursuit™ orNewpath™ (imazethapyr:(±)-2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-5-ethyl-3-pyridinecarboxylicacid); Scepter™ (imazaquin:2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-3-quinolinecarboxylicacid); Accent™ (nicosulfuron: 2-(((((4,6-dimethoxypyrimidin-2-yl)aminocarbonyl)) aminosulfonyl))-N,N-dimethyl-3-pyridinecarboxamide);Beacon™ (primisulfuron:3-[4,6-bis(difluoromethoxy)-pyrimidin-2-yl]-1-(2-methoxycarbonylphenylsulfonyl)urea); Raptor™ (imazamox:(+)-5-methoxymethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid; Cadre™ (imazapic:(±)-2-[4,5-dihydro-4-methyl-4-(1-methyl-ethyl)-5-oxo-1H-imidazol-2-yl]-5-methyl-3-pyridinecarboxylicacid; alternate chemical name(±)-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-5-methylnicotinicacid); Arsenal™ (imazapyr:2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-3-pyridinecarboxylicacid); Oust™ (sulfometuron methyl: chemical name2-[[[[(4,6-dimethyl-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]benzoicacid methyl ester); Ally™ (metsulfuron methyl: methyl2-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl]benzoate);Harmony™ (mixture of thifensulfuron methyl and tribenuron methyl:mixture of methyl-3-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl]-2-thiophenecarboxylate andmethyl-2-[[[[N-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)methylamino]carbonyl]amino]sulfonyl]benzoate); Pinnacle™ (thifensulfuronmethyl: methyl-3-[[[[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)amino]carbonyl]amino]sulfonyl]-2-thiophenecarboxylate); Glean™ or Telar™(chlorsulfuron:2-chloro-N-[(4-methoxy-6-methyl-1,3,5-triazin-2-yl)aminocarbonyl]benzenesulfonamide);Classic™ (chlorimuron ethyl: ethyl2-[[[[(4-chloro-6-methoxypyrimidin-2-yl)amino]carbonyl]amino]sulfonyl]benzoate);Express™ (tribenuron methyl: methyl2-[[[[N-(4-methoxy-6-methyl-1,3,5-triazin-2-yl)methylamino]carbonyl]amino]sulfonyl]benzoate);Assert™ (imazamethabenz methyl: m-toluic acid,6-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-, methyl ester; andp-toluic acid, 2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-, methylester); and Amber™ (triasulfuron:3-(6-methoxy-4-methyl-1,3,5-triazin-2-yl)-1-[2-(2-chloroethoxy)-phenylsulfonyl]-urea);Staple™ (pyrithiobac sodium: sodium 2-chloro-6-[(4,6-dimethoxypyrimidin-2-yl)thio]benzoate); and Matrix™ (rimsulfuron:N-((4,6-dimethoxypyrimidin-2-yl)aminocarbonyl)-3-(ethylsulfonyl)-2-pyridinesulfonamide).

Note on amino acid numbering convention used in the specification andclaims: As used in the Claims, and as used in the specification unlesscontext clearly indicates otherwise, the amino acids of rice AHAS arenumbered as corresponding to the numbering shown for the wild-type(Cypress) AHAS sequence (SEQ ID NO 17), or the corresponding position inother AHAS coding sequences where variations exist (such as were seenhere for the variety Kinmaze). In particular, amino acid position 627 isthe amino acid corresponding to amino acid 627 in SEQ ID NO 17, which isthe serine residue near the carboxy terminus in the wild type rice AHAS.Nucleotide positions are generally indicated indirectly, by reference tothe number of the amino acid encoded by a particular codon. For example,the codon in the wild-type (Cypress) AHAS coding sequence thatcorresponds to amino acid 627 is the AGT codon appearing in SEQ ID NO 14at nucleotides 1879-1881. By contrast, in the wild-type (Kinmaze)sequence of SEQ ID NO 2, this codon appears at nucleotides 1926-1928.Amino acid positions and corresponding nucleotide positions for otherspecies are determined by homology—for example, the amino acid or codonthat is in the position that is homologous to amino acid 627 (wild typeserine) in rice, or to the corresponding codon. Such homology may bedetermined through software commonly used in the art, such as GAP,PILEUP, or BLAST.

1. A herbicide-resistant rice plant, wherein said rice plant expressesan acetohydroxyacid synthase in which the amino acid at position 628 isglutamic acid.